Transformation system in the field of filamentous fungal hosts

ABSTRACT

A novel transformation system in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides is described. The invention also covers a process for producing large amounts of polypeptide or protein in an economical manner. The system comprises a transformed or transfected fungal strain of the genus  Chrysosporium , more particularly of  Chrysosporium lucknowense  and mutants or derivatives thereof. It also covers transformants containing  Chrysosporium  coding sequences, as well expression-regulating sequences of  Chrysosporium  genes. Also provided are novel fungal enzymes and their encoding sequences and expression-regulating sequences.

REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/047,709, filed Mar. 13, 2008, now U.S. Pat. No. 8,268,585, which is acontinuation of U.S. application Ser. No. 10/394,568, filed Mar. 21,2003, now U.S. Pat. No. 7,399,627, which is a continuation of U.S.application Ser. No. 09/548,938, filed Apr. 13, 2000, now U.S. Pat. No.6,573,086, which is a continuation-in-part of international applicationPCT/NL99/00618, filed Oct. 6, 1999, which is a continuation-in-part ofinternational application PCT/EP98/06496, filed Oct. 6, 1998, all ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A number of hosts for gene expression and methods of transformation havebeen disclosed in the prior art. Bacteria are often mentioned e.g.Escherichia coli. E. coli is however a micro-organism incapable ofsecretion of a number of proteins or polypeptides and as such isundesirable as host cell for production of protein or polypeptide at theindustrial level. An additional disadvantage for E. coli, which is validalso for bacteria in general, is that prokaryotes cannot provideadditional modifications required for numerous eukaryotic proteins orpolypeptides to be produced in an active form. Glycosylation of proteinsand proper folding of proteins are examples of processing required toensure an active protein or polypeptide is produced. To ensure suchprocessing one can sometimes use mammalian cells; however, thedisadvantage of such cells is that they are often difficult to maintainand require expensive media. Such transformation systems are thereforenot practical for production of proteins or polypeptides at theindustrial level. They may be cost efficient for highly pricedpharmaceutical compounds requiring relatively low amounts, but certainlynot for industrial enzymes.

A number of fungal expression systems have been developed e.g.Aspergillus niger, Aspergillus awamori, Aspergillus nidulans,Trichoderma reesei. A number of others have been suggested but forvarious reasons have not found wide-spread acceptance or use. In generalterms the ideal host must fulfill a large number of criteria:

-   -   The ideal host must be readily fermented using inexpensive        medium.    -   The ideal host should use the medium efficiently.    -   The ideal host must produce the polypeptide or protein in high        yield, i.e. must exhibit high protein to biomass ratio.    -   The ideal host should be capable of efficient secretion of the        protein or polypeptide.    -   The ideal host must enable ease of isolation and purification of        the desired protein or polypeptide.    -   The ideal host must process the desired protein or polypeptide        such that it is produced in an active form not requiring        additional activation or modification steps.    -   The ideal host should be readily transformed.    -   The ideal host should allow a wide range of expression        regulatory elements to be used thus ensuring ease of application        and versatility.    -   The ideal host should allow use of easily selectable markers        that are cheap to use. The ideal host should produce stable        transformants.    -   The ideal host should allow cultivation under conditions not        detrimental to the expressed protein or polypeptide e.g. low        viscosity, low shear.

Fungal systems that have not yet found widespread use are described e.g.in U.S. Pat. No. 5,578,463 by Berka et al suggesting Neurospora,Podospora, Endothia, Mucor, Cochoibolus and Pyricularia together withAspergillus and Trichoderma. However only illustrations oftransformation and expression are provided for Aspergillus andTrichoderma and no details are provided for any of the other suggestedhosts.

WO 96/02563 and U.S. Pat. Nos. 5,602,004, 5,604,129 and 5,695,985 toNovo Nordisk describe the drawbacks of Aspergillus and Trichodermasystems and suggests cultivation conditions for other fungi may be moresuited to large scale protein production. The only examples provided forany transformed cultures are those of Myceliophthora thermophile,Acremonium alabamense, Thielavia terrestris and Sporotrichumcellulophilum strains. The Sporotrichum strain is reported to lyse andproduce green pigment under fermentation conditions not leading to suchresults for the other strains. A non-sporulating mutant of Thielaviaterrestris is described as being the organism of choice by virtue of itsmorphology. However it is also stated that the protoplasting efficiencyof Thielavia and Acremonium (whereby the Acremonium strain used was theimperfect state of the Thielavia strain used) is low and that hygromycinwas not useful as a selection marker. A large number of others aresuggested as being potentially useful by virtue of their morphology butno transformation thereof is described. The suggested strains areCorynascus, Thermoascus, Chaetomium, Ctenomyces, Scytalidium andTalaromyces. The transformed hosts are mentioned as only producing lowlevels of the introduced Humicola xylanase with Thielavia producing thelowest amount; however, the information is ambiguous and could actuallyinfer Thielavia was the best embodiment. The nomenclature of thisreference is based on the ATCC names of Industrial Fungi of 1994. Thusit is apparent no high degree of heterologous expression was achievedand in fact no positive correlation could be derived between thepostulated morphology and the degree of expression. If any correlationcould be made, it was more likely to be negative. According to the 1996ATCC fungal classification Sporotrichum thermophilum ATCC 20493 is aMyceliophthora thermophila strain. Currently the strain is stillidentified as Myceliophthora thermophila. The unpredicatability of theart is apparent from these recent disclosures.

Also Allison et al (Curr. Genetics 21:225-229, 1992) describedtransformation of Humicola grisea var. thermoidea using the lithiumacetate method and a Humicola enzyme-encoding sequence, but no report ofexpression of heterologous protein from such a strain has been provided.

In 1997 a patent issued to Hawaii Biotechnology Group for transformedNeurospora for expression of mammalian peptide such as chymosin. Thetransformation of auxotrophic Neurospora crassa occurred withspheroplasts. Endogenous transcriptional regulatory regions wereintroduced and cotransformation was carried out. Nothing is mentionedconcerning other hosts and other transformation protocols. Nothing isapparent from the disclosure concerning the degree of expression. It isdoubtful whether the degree of expression is high, as immunotechniques(which are useful for detecting small amounts of protein) are the onlytechniques used to illustrate the presence of the protein. No actualisolation of the protein is disclosed.

WO 97/26330 of Novo Nordisk suggests a method of obtaining mutants offilamentous fungal parent cells having an improved property forproduction of heterologous polypeptide. The method comprises firstfinding a specific altered morphology followed by assessing whether atransformant produces more heterologous polypeptide than the parent. Themethod is illustrated only for strains of Fusarium A3/5 and Aspergillusoryzae. The method is suggested to be applicable for Aspergillus,Trichoderma, Thielavia, Fusarium, Neurospora, Acremonium, Tolyplocadium,Humicola, Scytalidium, Myceliophthora or Mucor. As stated above theunpredictability in the art and also the unpredictability of the methodof the cited application do not provide a generally applicable teachingwith a reasonable expectation of success.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to a novel transformation system in thefield of filamentous fungal hosts for expressing and secretingheterologous proteins or polypeptides. The invention also covers aprocess for producing large amounts of polypeptide in an economicalmanner. The system comprises a transformed or transfected fungal strainof the genus Chrysosporium, more particularly of Chrysosporiumlucknowense and mutants or derivatives thereof. It also coverstransformants containing Chrysosporium coding sequences. Novel mutantChrysosporium strains are disclosed as are novel enzymes derivedtherefrom. The subject invention further relates to novel enzymesderived from filamentous fungi, especially from strains of the genusChrysosporium, and to coding sequences and expression-regulatingsequences for these enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot as described in the Examples

FIG. 2 is a pUT720 map

FIG. 3 is a pUT970G map

FIG. 4 is a pUT1064 map

FIG. 5 is a pUT1065 map

FIG. 6 is a pF6g map

FIG. 7 is a pUT150 map

FIG. 8 is a pUT1152 map

FIG. 9 is a pUT1155 map

FIG. 10 is a pUT1160 map

FIG. 11 is a pUT1162 map

FIG. 12: Ion exchange chromatography on Macro Prep Q of non-boundfraction after DEAE-Toyopearl of F-60-31 CF sample.

FIG. 13A-E: pH dependencies of activity of enzymes from non-boundfractions of F-60-31 CF sample.

FIG. 14A-E: Stability of enzymes from non-bound fraction of F-60-31 CFsample at pH 5.5 and 7.5 (60° C.).

FIG. 15A-B: pH stability at 60° C. and 50° C. of 60 kD Xyl (pI 4.7) fromnon-bound fraction of F-60-31 sample.

FIG. 16A-E: Temperature dependencies of enzymes from non-bound fractionof F-60-31 sample.

FIG. 17: Ion exchange chromatography on Macro Prep Q of bound fractions50-53 after DEAE-Toyopearl of F-60-8 sample.

FIG. 18A-B: pH and temperature dependencies of .alpha.-galactosidaseactivity of F-60-43, UF-conc.

FIG. 19: pH dependencies of activity of 48 kD CBH (pI 4.4) from boundfractions of F-60-8, UF-conc.

FIG. 20: Temperature dependencies of activity towards p-nitrophenylbutyrate of F-60-8 UF-conc.

FIG. 21: pH dependencies of activity towards p-nitrophenyl butyrate ofF-60-8 UF-conc.

FIG. 22: pH courses of activities of 30 kD (pI 8.9) and 90 kD (pI 4.2)proteases toward C1 proteins (50° C., 30 min. incubation).

FIG. 23A-D: Effect of 30 kD (pI 8.9) “alkaline” protease on xylanaseactivity of the non-bound-fraction (Macro Prep Q™) of F 60-31 CF at 50°C.

FIG. 24A-F: Effect of 90 kD (pI 4.2) “neutral” protease on CMCaseactivity of the proteins in the bound fraction #44-45 (DEAE-Toyopearl)of F 60-8 UV-conc sample at 50° C.

FIG. 25: Complete hydrolysis of polygalacturonic acid by 65 kDpolygalacturonase (pI 4.4): 50° C., pH 4.5; concentration of PGA=5 g/l,concentration of protein=0.1 g/l.

FIG. 26A-B: pH- and temperature dependencies of polygalacturonaseactivity of F-60-43 UF-conc.

FIG. 27: Inhibition of activity toward MUF-cellobioside by cellobiosefor 55 kD CBH (pI 4.4): pH 4.5, 40 C.

FIG. 28: Synergistic effect between 25 kD Endo (pI 4.1) and 55 kD CBH(pI 4.4) toward avicel (40 C, pH 5, 25 min).

FIG. 29A-D: Complete hydrolysis of CMC (a and c) and avicel (b and d) bythe enzymes isolated from bound fractions of F-60-8 UF-conc. sample (50°C., pH 5): concentration of CMC and avicel=5 g/l, concentration of 25 kDEndo=0.01 g/l, concentration of 43 kD Endo=0.02 g/l; (a and b) 25 kDEndo (pI 4.1), (c and d) 43 kD Endo (pI 4.2).

FIG. 30A-D: Complete hydrolysis of CMC (a and b) and avicel (c and d) by55 kD CBH (pI 4.4) without (a and c) and with (b and d)glucono-.delta.-lactone (50° C., pH 4.5): concentration of CMC andavicel=5 g/l, concentration of protein=0.1 g/l, concentration ofglucono-.delta.-lactone=5 g/l.

FIG. 31A-B: pH-Dependence of CMCase and RBB-CMCase activities of theenzymes isolated from F-60-8 UF-conc. sample: (a) 25 kD Endo (pI 4.1),(b) 43 kD Endo (pI 4.2).

FIG. 32: pH-Dependencies of CMCase and RBB-CMCase activities of 55 kDCBH (pI 4.4).

FIG. 33A-C: Temperature dependencies of CMCase activity (pH 4.5) of theenzymes isolated from bound fractions of F-60-8 UF-conc. sample: (a) 55kD CBH (pI 4.4), (b) 25 kD Endo (pI 4.1), (c) 43 kD Endo (pI 4.2).

FIG. 34A-C: pH-stability (50° C.) of the enzymes isolated from boundfractions of F-60-8 UF-conc. sample: (a) 55 kD CBH (pI 4.4), (b) 25 kDEndo (pI 4.1), (c) 43 kD Endo (pI 4.2).

FIG. 35: Adsorption of the enzymes isolated from bound fractions ofF-60-8 UF-conc. sample.

FIG. 36: Purification scheme of F-60-8 (UF-conc) and F-60-31 CF Samplesfor C1 Protein Isolation.

DETAILED DESCRIPTION OF THE INVENTION

We now describe an alternative fungal expression system with thesimplicity of use of the above-mentioned Aspergilli and Trichodermafulfilling the above requirements. The new system has not been taught orsuggested in the prior art. The new system according to the inventionprovides the additional advantages that transformation rates are higherthan those for the frequently used Trichoderma reesei system. Inaddition the culture conditions offer the additional bonus of beingadvantageous for the expressed polypeptide.

We further describe a number of industrially interesting enzymes derivedfrom the novel expressing system, together with full sequenceinformation. We also describe novel promoter systems derived fromChrysosporium strains and useful for expressing homologous andheterologous genes.

The present invention is thus also concerned with glycosyl hydrolases ofthe families 7 (e.g. cellobiohydrolases), 10 (e.g. xylanases) and 12(e.g. endoglucanases), and glyceraldehyde phosphate dehydrogenases, asidentified by their amino acid sequence, as well as peptides derivedfrom these enzymatic proteins, and with nucleic acid sequences encodingthese peptides and proteins, as well as, in particular, with regulatingsequences related to these genes.

In particular, the present invention pertains to isolated or recombinantenzymic proteins or active parts thereof of the four classes referred toabove, including mutants thereof having at least a certain degree ofsequence identity as specified in the further disclosure and in theclaims, as well as nucleic acid sequences encoding these proteins orparts thereof, and/or nucelic acid sequences regulating theirexpression. These enzymes are especially: (1) a glycosyl hydrolase offamily 7 (cellobiohydrolase, CBH1) having at least 75%, preferably atleast 80% or even at least 85% amino acid identity with the sequence ofSEQ ID No 1; (2) a glycosyl hydrolase of family 10 (endoxylanase XYLF orXYL1) having at least 70%, preferably at least 75% or even at least 80%amino acid identity with the sequence of SEQ ID No 2; (3) a glycosylhydrolase family of 12 (endoglucanase, EG3) having at least 65%,preferably at least 70% or even at least 80% amino acid identity withthe sequence of SEQ ID No. 3; and (4) a glyceraldehyde phosphatedehydrogenase (GPD1) having at least 86%, preferably at least 90% oreven at least 93% amino acid identity with the sequence of SEQ ID No 4.Polypeptides and nucleic acid sequences encoding these polypeptides,having at least 20, preferably at least 30 contiguous amino acids of SEQID No's 1-4 are also a preferred part of the invention.

The recombinant enzymes may comprise essentially the complete protein,or a truncated protein having at least part of the enzymatic activity.Such truncated part may be the catalytic domain, or at least about 75%of the amino acids thereof. By way of example, the catalytic domain ofthe CBH1 according to the invention comprises the aminoacids 20-495 ofthe aminoacid sequence of SEQ ID No. 1, and the catalytic domain of theXYL1 according to the invention comprises the aminoacids 54-384 of theaminoacid sequence of SEQ ID No. 2. The catalytic domain may or may notbe combined with a signal sequence originating from another proteinand/or with a carbohydrate-binding domain from another enzymic protein.Alternatively, the cellulose-binding domain of the enzymes of theinvention (CBH1 and XYL1) may be fused to catalytic domains of otherenzymic proteins.

The nucleic acid sequences according to of the invention may be completeprotein-encoding regions or oligonucleotides or, preferentially,expression-regulating sequences. Oligonucleotides may be used also asprobes for identifying genes corresponding to, but not identical to thegenes of SEQ ID No.'s 1-4; these genes, when fulfilling the percentageidentity criteria defined herein, as well as encoding and non-encodingparts thereof and their expression products are also part of theinvention.

The invention also pertains to expression systems (cassettes) comprisingeither an expression-regulating region (including a promoter) of any ofthe four protein classes fused to a gene encoding another protein ofinterest, or an encoding region of any of these proteins fused toanother expression regulating region, or both the expression-regulatingregion and the protein-encoding region of these novel proteins. Theexpression-regulating region comprises at least 60%, preferably at least70%, more preferably at least 75% or even 80% of the 5′-non-codingregion of SEQ ID No.'s 1-4, and/or at least 20, especially at least 40contiguous nucleotides from these 5′ non-coding regions. Terminatingsequences similarly derived from the 3′ non-coding regions of the genesof the invention are also useful in expressing cassettes, whethercombined with homologous or heterologous genes.

These expression systems may be contained in a Chrysosporium host, suchas a Chrysosporium lucknowense host, or in another non-fungal or,preferably, fungal host. Examples of other fungal hosts are otherChrysosporium species or strains, Fusarium species, Aspergillus speciesetc. Such host may be advantageously a host that does not itself,intrinsically or as a result of the culture conditions, produce aprotein corresponding to the protein of interest, so as to simplify therecovery of the protein of interest.

Where reference is made in this specification and in the appendingclaims to “polypeptides” or “peptides” or “polypeptides of interest” or“peptides of interest” as the products of the expression system of theinvention, this term also comprise proteins, i.e. polypeptides having aparticular function and/or secondary and/or tertiary structure. Wherereference is made to percentage amino acid identity, such identityrelates to e complete protein or a to a specific part defined by initialand final amino acid number, as determined by the conventionally usedBLAST algorithm.

In the production method of the invention, the pH of the culture mediumcan be neutral or alkaline thus no longer subjecting the producedprotein or polypeptide to aggressive and potentially inactivating acidpH. It is also possible to culture at acid pH such as pH 4 for caseswhere the protein or polypeptide is better suited to an acidicenvironment. Suitably culture can occur at a pH between 4.0-10.0. Apreference however exists for neutral to alkaline pH as the host strainexhibits better growth at such pH, e.g. between 6 and 9. Growth atalkaline pH which can be from pH 8 up and can even be as high as 10 isalso a good alternative for some cases. Also the cultivation temperatureof such host strains is advantageous to the stability of some types ofproduced polypeptide. The cultivation temperature is suitably at atemperature of 25-43° C. A temperature in the range from 40° C. down to23° C. or 30° C. is also advantageously applied. Clearly such conditionsare of particular interest for production of mammalian polypeptides. Theselected temperature will depend on cost effectiveness of thecultivation and sensitivity of the polypeptide or cultivation strain.The conditions will be determined by the skilled person without undueburden on a case-by-case basis, as is common in the art.

It has also been ascertained that the biomass and viscosity relations tothe amount of protein produced is exceedingly favourable for the hostaccording to the invention. Comparisons have been carried out withTrichoderma longibrachiatum (formerly also known as Trichoderma reesei)and with Aspergillus niger. Trichoderma longibrachiatum gave 2.5-5 g/lbiomass, Aspergillus niger gave 5-10 g/l biomass and the host accordingto the invention gave 0.5-1 g/l biomass under their respective optimisedconditions. This thus offers 5-10 fold improvement over the commerciallyused strains. These commercial strains are strains which themselves areconsidered in the art to be high producers of proteins and they aresuccessfully used for commercial protein production. They have beencultured under their optimal conditions developed and run viably inlarge-scale commercial fermenters. The same strains were used toillustrate enormous improvement in viscosity values for cultures of thehost according to the invention. At the end of the fermentation processTrichoderma longibrachiatum gave a value of 200-600 cP (Centipoise),Aspergillus niger gave a value of 1500-2000 cP and the host according tothe invention gave a value below 10 cP. This thus provides at least20-200 fold improvement for viscosity values over the commercially usedstrains. A quite surprising further aspect was that the protein levelsdetermined for the host cells according to the invention were muchhigher than for the commercial Aspergilli and Trichoderma reeseistrains, even with the above mentioned surprisingly low biomass andviscosity levels. In summary an easy to use versatile improvedtransformation system and expression system with improved culturingconditions has hereby been introduced. The strains according to theinvention produce surprisingly higher protein levels under theseimproved conditions and in addition they do such in a shorter fermentertime.

The subject invention is directed at mutant Chrysosporium strainscomprising a nucleic acid sequence encoding a heterologous protein orpolypeptide, said nucleic acid sequence being operably linked to anexpression regulating region and optionally a secretion signal encodingsequence and/or a carrier protein encoding sequence. Preferably arecombinant strain according to the invention will secrete thepolypeptide of interest. This will avoid the necessity of disrupting thecell in order to isolate the polypeptide of interest and also minimisethe risk of degradation of the expressed product by other components ofthe host cell.

Chrysosporium can be defined by morphology consistent with thatdisclosed in Barnett and Hunter 1972, Illustrated Genera of ImperfectFungi, 3rd Edition of Burgess Publishing Company. Other sourcesproviding details concerning classification of fungi of the genusChrysosporium are known e.g. Sutton Classification (Van Oorschot, C. A.N. (1980) “A revision of Chrysosporium and allied genera” in Studies inMycology No. 20 of the CBS in Baarn The Netherlands p1-36). CBS is oneof the depository institutes of the Budapest Treaty. According to theseteachings the genus Chrysosporium falls within the family Moniliaceaewhich belongs to the order Hyphomycetales. The criteria that can be usedare the following:

1. Signs of Hphomycetales order:

Conidia are produced directly on mycelium, on separate sporogenous cellsor on distinct conidiophores.

2. Signs of Moniliaceae family:

Both conidia and conidiophores (if present) are hyaline or brightlycoloured; conidiophores are single or in loose clusters.

3. Signs of Chrysosporium Corda 1833 genus:

Colonies are usually spreading, white, sometimes cream-coloured, palebrown or yellow, felty and/or powdery. Hyphae are mostly hyaline andsmooth-walled, with irregular, more or less orthotopic branching.Fertile hyphae exhibit little or no differentiation. Conidia areterminal and lateral, thallic, borne all over the hyphae, sessile or onshort protrusions or side branches, subhyaline or pale yellow, thin- orthick-walled, subglobose, clavate, pyriform, orobovoid, 1-celled, rarely2-celled, truncate. Intercalary conidia are sometimes present, aresolitary, occasionally catenate, subhyaline or pale yellow, broader thanthe supporting hyphae, normally 1-celled, truncate at both ends.Chlamydospores are occasionally present.

Another source providing information on fungal nomenclature is ATCC(US). Their website is may be accessed on the World Wide Web (HTTPprotocol) at atcc.org. CBS also has a website located on the World WideWeb (HTTP protocol) at cbs.knaw.nl providing relevant information. VKMin Moscow is also a reliable source of such information, located on theWorld Wide Web (HTTP protocol) at bdt.org.br.bdt.msdn.vkm/general.Another source is the United States Department of Agriculture ResearchService website (located on an NT webserver rather than the World WideWeb) at ars-grin.gov/fungaldatabases. All these institutions can provideteaching on the distinguishing characteristics of a Chrysosporium.

Strains defined as being of Myceliophthora thermophile are notconsidered to define Chrysosporium strains according to the definitionof the invention. In the past there has been considerable confusion overthe nomenclature of some Myceliophthora strains. Preferably theChrysosporium according to the invention are those which are clearlydistinguishable as such and cannot be confused with Myceliophthora,Sporotrichum or Phanerochaete Chrysosporium.

The following strains are defined as Chrysosporium but the definition ofChrysosporium is not limited to these strains: C. botryoides, C.carmichaelii, C. crassitunicatum, C. europae, C. evolceannui, C.farinicola, C. fastidium, C. filiforme, C. georgiae, C. globiferum, C.globiferum var. articulatum, C. globiferum var. niveum, C. hirundo, C.hispanicum, C. holmii, C. indicum, C. ihops, C. keratinophilum, C.kreiselii, C. kuzurovianum, C. lignorum, C. lobatum, C. lucknowense, C.lucknowense Garg 27K, C. medium, C. medium var. spissescens, C.mephiticum, C. merdarium, C. merdarium var. roseum, C. minor, C.pannicola, C. parvum, C. parvum var. crescens, C. pilosum, C.pseudomerdarium, C. pyriformis, C. queenslandicum, C. sigleri, C.sulfureum, C. synchronum, C. tropicum, C. undulatum, C. vallenarense, C.vespertilium, C. zonatum.

C. lucknowense forms one of the species of Chrysosporium that haveraised particular interest as it has provided a natural high producer ofcellulase proteins (WO 98/15633 and related U.S. Pat. No. 5,811,381, aswell as U.S. Pat. No. 6,015,707). The characteristics of thisChrysosporium lucknowense are:

Colonies attain 55 mm diameter on Sabouraud glucose agar in 14 days, arecream-coloured, felty and fluffy; dense and 3-5 mm high; margins aredefined, regular, and fimbriate; reverse pale yellow to cream-coloured.Hyphae are hyaline, smooth- and thin-walled, little branched. Aerialhyphae are mostly fertile and closely septate, about 1-3.5 μm wide.Submerged hyphae are infertile, about 1-4.5 μm wide, with the thinnerhyphae often being contorted. Conidia are terminal and lateral, mostlysessile or on short, frequently conical protrusions or short sidebranches. Conidia are solitary but in close proximity to one another,1-4 conidia developing on one hyphal cell, subhyaline, fairly thin- andsmooth-walled, mostly subglobose, also clavate orobovoid, 1-celled,2.5-11.times.1.5-6 μm, with broad basal scars (1-2 μm). Intercalaryconidia are absent. Chlamydospores are absent. ATCC 44006, CBS 251.72,CBS 143.77 and CBS 272.77 are examples of Chrysosporium lucknowensestrains and other examples are provided in WO 98/15633 and U.S. Pat. No.5,811,381.

A further strain was isolated from this species with an even higherproduction capacity for cellulases. This strain is called C1 by itsinternal notation and was deposited with the International Depository ofthe All Russian Collection of micro-organisms of the Russian Academy ofSciences Bakrushina Street 8, Moscow, Russia 113184 on Aug. 29, 1996, asa deposit according to the Budapest Treaty and was assigned AccessionNumber VKM F-3500D. It is called Chrysosporium lucknowense Garg 27K. Thecharacteristics of the C1 strain are as follows:

Colonies grow to about 55-66 mm diameter in size on potato-dextrose agarin about 7 days; are white-cream-coloured, felty, 2-3 μm high at thecenter; margins are defined, regular, fimbriate; reverse pale,cream-coloured. Hyphae are hyaline, smooth- and thin-walled, littlebranched. Aerial hyphae are fertile, septate, 2-3 mm wide. Submergedhyphae are infertile. Conidia are terminal and lateral; sessile or onshort side branches; absent; solitary, but in close proximity to oneanother, hyaline, thin- and smooth-walled, subglobose, clavate orobovoid, 1-celled, 4-10 μm. Chlamydospores are absent. Intercalaryconidia are absent.

The method of isolation of the C1 strain is described in WO 98/15633 andU.S. Pat. No. 5,811,381. Strains exhibiting such morphology are includedwithin the definition of Chrysosporium according to the invention. Alsoincluded within the definition of Chrysosporium are strains derived fromChrysosporium predecessors including those that have mutated somewhateither naturally or by induced mutagenesis. In particular the inventioncovers mutants of Chrysosporium obtained by induced mutagenis,especially by a combination of irradiation and chemical mutagenesis.

For example strain C1 was mutagenised by subjecting it to ultravioletlight to generate strain UV 13-6. This strain was subsequently furthermutated with N-methyl-N′-nitro-N-nitrosoguanidine to generate strainNG7C-19. The latter strain in turn was subjected to mutation byultraviolet light resulting in strain UV18-25. During this mutationprocess the morphological characteristics have varied somewhat inculture in liquid or on plates as well as under the microscope. Witheach successive mutagenesis the cultures showed less of the fluffy andfelty appearance on plates that are described as being characteristic ofChrysosporium, until the colonies attained a flat and matted appearance.A brown pigment observed with the wild type strain in some media wasalso less prevalent in mutant strains. In liquid culture the mutantUV18-25 was noticeably less viscous than the wild type strain C1 and themutants UV13-6 and NG7C-19. While all strains maintained the grossmicroscopic characteristics of Chrysosporium, the mycelia becamenarrower with each successive mutation and with UV18-25 distinctfragmentation of the mycelia could be observed. This mycelialfragmentation is likely to be the cause of the lower viscosityassociated with cultures of UV18-25. The ability of the strains tosporulate decreased with each mutagenic step. The above illustrates thatfor a strain to belong to the genus Chrysosporium there is some leewayfrom the above morphological definition. At each mutation stepproduction of cellulase and extracellular proteins has in addition alsoincreased, while several mutations resulted in decrease of proteaseexpression. Criteria with which fungal taxonomy can be determined areavailable from CBS, VKMF and ATCC for example.

In particular the anamorph form of Chrysosporium has been found to besuited for the production application according to the invention. Themetabolism of the anamorph renders it extremely suitable for a highdegree of expression. A teleomorph should also be suitable as thegenetic make-up of the anamorphs and teleomorphs is identical. Thedifference between anamorph and teleomorph is that one is the asexualstate and the other is the sexual state. The two states exhibitdifferent morphology under certain conditions.

It is preferable to use non-toxic Chrysosporium strains of which anumber are known in the art as this will reduce risks to the environmentupon large scale production and simplify production procedures with theconcomitant reduction in costs.

An expression-regulating region is a DNA sequence recognised by the hostChrysosporium strain for expression. It comprises a promoter sequenceoperably linked to a nucleic acid sequence encoding the polypeptide tobe expressed. The promoter is linked such that the positioning vis-a-visthe initiation codon of the sequence to be expressed allows expression.The promoter sequence can be constitutive or inducible. Any expressionregulating sequence or combination thereof capable of permittingexpression of a polypeptide from a Chrysosporium strain is envisaged.The expression regulating sequence is suitably a fungalexpression-regulating region e.g. an ascomycete regulating region.Suitably the fungal expression regulating region is a regulating regionfrom any of the following genera of fungi: Aspergillus, Trichoderma,Chrysosporium (preferred), Hansenula, Mucor, Pichia, Neurospora,Tolypocladium, Rhizomucor, Fusarium, Penicillium, Saccharomyces,Talaromyces or alternative sexual forms thereof like Emericella,Hypocrea e.g. the cellobiohydrolase promoter from Trichoderma,glucoamylase promoter from Aspergillus, glyceraldehyde phosphatedehydrogenase promoter from Aspergillus, alcohol dehydrogenase A andalcohol dehydrogenase R promoter of Aspergillus, TAKA amylase promoterfrom Aspergillus, phosphoglycerate and cross-pathway control promotersof Neurospora, aspartic proteinase promoter of Rhizomucor miehei, lipasepromoter of Rhizomucor miehei and beta-galactosidase promoter ofPenicillium canescens. An expression regulating sequence from the samegenus as the host strain is extremely suitable, as it is most likely tobe specifically adapted to the specific host. Thus preferably theexpression regulating sequence is one from a Chrysosporium strain.

We have found particular strains of Chrysosporium to express proteins inextremely large amounts and natural expression regulating sequences fromthese strains are of particular interest. These strains are internallydesignated as Chrysosporium strain C1, strain UV13-6, strain NG7C-19 andstrain UV18-25. They have been deposited in accordance with the BudapestTreaty with the All Russian Collection (VKM) depository institute inMoscow. Wild type C1 strain was deposited in accordance with theBudapest Treaty with the number VKM F-3500 D, deposit date Aug. 29,1996, C1 UV13-6 mutant was deposited with number VKM F-3632 D, anddeposit date Sep. 20, 1998, C1 NG7C-19 mutant was deposited with numberVKM F-3633 D and deposit date Sep. 20, 1998 and C1 UV18-25 mutant wasdeposited with number VKM F-3631 D and deposit date Sep. 2, 1998.

Preferably an expression-regulating region enabling high expression inthe selected host is applied. This can also be a highexpression-regulating region derived from a heterologous host, such asare well known in the art. Specific examples of proteins known to beexpressed in large quantities and thus providing suitable expressionregulating sequences for the invention are without being limited theretohydrophobin, protease, amylase, xylanase, pectinase, esterase,beta-galactosidase, cellulase (e.g. endo-glucanase, cellobiohydrolase)and polygalacturonase. The high production has been ascertained in bothsolid state and submerged fermentation conditions. Assays for assessingthe presence or production of such proteins are well known in the art.The catalogues of Sigma and Megazyme for example provide numerousexamples. Megazyme is located at Bray Business Park, Bray, CountyWicklow in Ireland. Sigma Aldrich has many affiliates world wide e.g.USA P.O. Box 14508 St. Louis Mo. For cellulase we refer to commerciallyavailable assays such as CMCase assays, endoviscometric assays,Avicelase assays, beta-glucanase assays, RBBCMCase assays, Cellazyme Cassays. Xylanase assays are also commercially available (e.g. DNS andMegazyme). Alternatives are well known to a person skilled in the artand can be found from general literature concerning the subject and suchinformation is considered incorporated herein by reference. By way ofexample we refer to “Methods in Enzymology Volume 1, 1955 right throughto volumes 297-299 of 1998. Suitably a Chrysosporium promoter sequenceis applied to ensure good recognition thereof by the host.

We have found that heterologous expression-regulating sequences work asefficiently in Chrysosporium as native Chrysosporium sequences. Thisallows well known constructs and vectors to be used in transformation ofChrysosporium as well as offering numerous other possibilities forconstructing vectors enabling good rates of expression in this novelexpression and secretion host. For example standard Aspergillustransformation techniques can be used as described for example byChristiansen et al in Bio/Technol. 6:1419-1422 (1988). Other documentsproviding details of Aspergillus transformation vectors, e.g. U.S. Pat.Nos. 4,816,405, 5,198,345, 5,503,991, 5,364,770 and 5,578,463,EP-B-215.594 (also for Trichoderma) and their contents are incorporatedby reference. As extremely high expression rates for cellulase have beenascertained for Chrysosporium strains, the expression regulating regionsof such proteins are particularly preferred. We refer for specificexamples to the previously mentioned deposited Chrysosporium strains.

A nucleic acid construct comprising a nucleic acid expression regulatoryregion from Chrysosporium, preferably from Chrysosporium lucknowense ora derivative thereof forms a separate embodiment of the invention asdoes the mutant Chrysosporium strain comprising such operably linked toa gene encoding a polypeptide to be expressed. Suitably such a nucleicacid construct will be an expression regulatory region fromChrysosporium associated with cellulase or xylanase expression,preferably cellobiohydrolase expression, more specifically expression ofa 55 kDa cellobiohydrolase. The Chrysosporium promoter sequences of anendoglucanase of 25 kDa (C1-EG5) and of an endoglucanase of 43 kDa(C1-EG6), wherein the molecular weights are determined according to SDSPAGE (with the molecular weights according to amino acid sequence databeing 21.9 kDa and 39.5 kDa), are provided by way of example. Thus, theChrysosporium promoter sequences of hydrophobin, protease, amylase,xylanase, esterase, pectinase, beta-galactosidase, cellulase (e.g.endoglucanase, cellobiohydrolase) and polygalacturonase are consideredto also fall within the scope of the invention. Any of the promoters orregulatory regions of expression of enzymes disclosed in Table A or Bcan be suitably employed. The nucleic acid sequence according to theinvention can suitably be obtained from a Chrysosporium strain accordingto the invention, such strain being defined elsewhere in thedescription. The manner in which promoter sequences can be determinedare numerous and well known in the art. Nuclease deletion experiments ofthe region upstream of the ATG codon at the beginning of the relevantgene will provide such sequence. Also for example analysis of consensussequences can lead to finding a gene of interest. Using hybridisationand amplification techniques one skilled in the art can readily arriveat the corresponding promoter sequences.

The promoter sequences of C1 endoglucanases were identified in thismanner, by cloning the corresponding genes, and are given in SEQ IDNo.'s 5 (EG5) and 6 (EG6), respectively. Other preferred promotersaccording to the invention are the 55 kDa cellobiohydrolase (CBH1)promoter and the 30 kDa xylanase (Xy1F) promoters, as the enzymes areexpressed at high level by their own promoters. The correspondingpromoter sequences can be identified in a straightforward manner bycloning as described below for the endoglucanase promoters, using thesequence information given in SEQ ID No. 1 (for CBH1) and SEQ ID No. 2(for Xy1F), respectively. The promoters of the carbohydrate-degradingenzymes of Chrysosporium, especially C1 promoters, can advantageously beused for expressing desired polypeptides in a host organism, especiallya fungal or other microbial host organism. Promoter sequences having atleast 60%, preferably at least 70%, most preferably at least 80%nucleotide sequence identity with the sequence given in SEQ ID No's 1and 2, or with the sequences found for other Chrysosporium genes, arepart of the present invention.

For particular embodiments of the recombinant strain and the nucleicacid sequence according to the invention we also refer to the examples.We also refer for the recombinant strains to prior art describing highexpression promoter sequences in particular those providing highexpression in fungi e.g. such as are disclosed for Aspergillus andTrichoderma. The prior art provides a number of expression regulatingregions for use in Aspergillus e.g. U.S. Pat. No. 5,252,726 of Novo andU.S. Pat. No. 5,705,358 of Unilever. The contents of such prior art arehereby incorporated by reference.

The hydrophobin gene is a fungal gene that is highly expressed. It isthus suggested that the promoter sequence of a hydrophobin gene,preferably from Chrysosporium, may be suitably applied as expressionregulating sequence in a suitable embodiment of the invention.Trichoderma reesei and Trichoderma harzianum gene sequences forhydrophobin have been disclosed for example in the prior art as well asa gene sequence for Aspergillus fumigatis and Aspergillus nidulans andthe relevant sequence information is hereby incorporated by reference(Munoz et al, Curr. Genet. 1997, 32(3):225-230; Nakari-Setala T. et al,Eur. J. Biochem. 1996 15:235 (1-2):248-255, M. Parta et al, Infect.Immun. 1994 62 (10): 4389-4395 and Stringer M. A. et al. Mol. Microbiol.1995 16(1):33-44). Using this sequence information a person skilled inthe art can obtain the expression regulating sequences of Chrysosporiumhydrophobin genes without undue experimentation following standardtechniques as suggested already above. A recombinant Chrysosporiumstrain according to the invention can comprise a hydrophobin-regulatingregion operably linked to the sequence encoding the polypeptide ofinterest.

An expression regulating sequence can also additionally comprise anenhancer or silencer. These are also well known in the prior art and areusually located some distance away from the promoter. The expressionregulating sequences can also comprise promoters with activator bindingsites and repressor binding sites. In some cases such sites may also bemodified to eliminate this type of regulation. Filamentous fungalpromoters in which creA sites are present have been described. Such creAsites can be mutated to ensure the glucose repression normally resultingfrom the presence of the non-mutated sites is eliminated. Gist-Brocades'WO 94/13820 illustrates this principle. Use of such a promoter enablesproduction of the polypeptide encoded by the nucleic acid sequenceregulated by the promoter in the presence of glucose. The same principleis also apparent from WO 97/09438. These promoters can be used eitherwith or without their creA sites. Mutants in which the creA sites havebeen mutated can be used as expression regulating sequences in arecombinant strain according to the invention and the nucleic acidsequence it regulates can then be expressed in the presence of glucose.Such Chrysosporium promoters ensure derepression in an analogous mannerto that illustrated in WO 97/09438. The identity of creA sites is knownfrom the prior art. Alternatively, it is possible to apply a promoterwith CreA binding sites that have not been mutated in a host strain witha mutation elsewhere in the repression system e.g. in the creA geneitself, so that the strain can, notwithstanding the presence of creAbinding sites, produce the protein or polypeptide in the presence ofglucose.

Terminator sequences are also expression-regulating sequences and theseare operably linked to the 3′ terminus of the sequence to be expressed.Any fungal terminator is likely to be functional in the hostChrysosporium strain according to the invention. Examples are A.nidulans trpC terminator (1), A. niger alpha-glucosidase terminator (2),A. niger glucoamylase terminator (3), Mucor miehei carboxyl proteaseterminator (U.S. Pat. No. 5,578,463) and the Trichoderma reeseicellobiohydrolase terminator. Naturally Chrysosporium terminatorsequences will function in Chrysosporium and are suitable e.g. CBH1 orEG6 terminator.

A suitable recombinant Chrysosporium strain according to the inventionhas the nucleic acid sequence to be expressed operably linked to asequence encoding the amino acid sequence defined as signal sequence. Asignal sequence is an amino acid sequence which when operably linked tothe amino acid sequence of the expressed polypeptide allows secretionthereof from the host fungus. Such a signal sequence may be one normallyassociated with the heterologous polypeptide or may be one native to thehost. It can also be foreign to both host and the polypeptide. Thenucleic acid sequence encoding the signal sequence must be positioned inframe to permit translation of the signal sequence and the heterologouspolypeptide. Any signal sequence capable of permitting secretion of apolypeptide from a Chrysosporium strain is envisaged. Such a signalsequence is suitably a fungal signal sequence, preferably an ascomycetesignal sequence.

Suitable examples of signal sequences can be derived from yeasts ingeneral or any of the following specific genera of fungi: Aspergillus,Trichoderma, Chrysosporium, Pichia, Neurospora, Rhizomucor, Hansenula,Humicola, Mucor, Tolypocladium, Fusarium, Penicillium, Saccharomyces,Talaromyces or alternative sexual forms thereof like Emericella,Hypocrea. Signal sequences that are particularly useful are oftennatively associated with the following proteins a cellobiohydrolase, anendoglucanase, a beta-galactosidase, a xylanase, a pectinase, anesterase, a hydrophobin, a protease or an amylase. Examples includeamylase or glucoamylase of Aspergillus or Humicola (4), TAKA amylase ofAspergillus oryzae, alpha-amylase of Aspergillus niger, carboxylpeptidase of Mucor (U.S. Pat. No. 5,578,463), a lipase or proteinasefrom Rhizomucor miehei, cellobiohydrolase of Trichoderma (5),beta-galactosidase of Penicillium canescens and alpha mating factor ofSaccharomyces.

Alternatively the signal sequence can be from an amylase or subtilisingene of a strain of Bacillus. A signal sequence from the same genus asthe host strain is extremely suitable as it is most likely to bespecifically adapted to the specific host thus preferably the signalsequence is a signal sequence of Chrysosporium. We have found particularstrains of Chrysosporium to excrete proteins in extremely large amountsand naturally signal sequences from these strains are of particularinterest. These strains are internally designated as Chrysosporiumstrain C1, strain UV13-6, strain NG7C-19 and strain UV18-25. They havebeen deposited in accordance with the Budapest Treaty as describedelsewhere in this description. Signal sequences from filamentous fungi,yeast and bacteria are useful. Signal sequences of non-fungal origin arealso considered useful, particularly bacterial, plant and mammalian.

A recombinant Chrysosporium strain according to any of the embodimentsof the invention can further comprise a selectable marker. Such aselectable marker will permit easy selection of transformed ortransfected cells. A selectable marker often encodes a gene productproviding a specific type of resistance foreign to the non-transformedstrain. This can be resistance to heavy metals, antibiotics and biocidesin general. Prototrophy is also a useful selectable marker of thenon-antibiotic variety. Non-antibiotic selectable markers can bepreferred where the protein or polypeptide of interest is to be used infood or pharmaceuticals with a view to speedier or less complicatedregulatory approval of such a product. Very often the GRAS indication isused for such markers. A number of such markers are available to theperson skilled in the art. The FDA e.g. provides a list of such. Mostcommonly used are selectable markers selected from the group conferringresistance to a drug or relieving a nutritional defect e.g the groupcomprising amdS (acetamidase), hph (hygromycin phosphotransferase), pyrG(orotidine-5′-phosphate decarboxylase), trpC (anthranilate synthase),argB (ornithine carbamoyltransferase), sC (sulphate adenyltransferase),bar (phosphinothricin acetyltransferase), glufosinate resistance, niaD(nitrate reductase), a bleomycin resistance gene, more specifically Shble, sulfonylurea resistance e.g. acetolactate synthase mutation ilv1.Selection can also be carried out by virtue of cotransformation wherethe selection marker is on a separate vector or where the selectionmarker is on the same nucleic acid fragment as the polypeptide-encodingsequence for the polypeptide of interest.

As used herein the term heterologous polypeptide is a protein orpolypeptide not normally expressed and secreted by the Chrysosporiumhost strain used for expression according to the invention. Thepolypeptide can be of plant or animal (vertebrate or invertebrate)origin e.g. mammalian, fish, insect, or micro-organism origin, with theproviso it does not occur in the host strain. A mammal can include ahuman. A micro-organism comprises viruses, bacteria, archaebacteria andfungi i.e. filamentous fungi and yeasts. Bergey's Manual for BacterialDeterminology provides adequate lists of bacteria and archaebacteria.For pharmaceutical purposes quite often a preference will exist forhuman proteins thus a recombinant host according to the inventionforming a preferred embodiment will be a host wherein the polypeptide isof human origin. For purposes such as food production suitably theheterologous polypeptide will be of animal, plant or algal origin. Suchembodiments are therefore also considered suitable examples of theinvention. Alternative embodiments that are useful also include aheterologous polypeptide of any of bacterial, yeast, viral,archaebacterial and fungal origin. Fungal origin is most preferred.

A suitable embodiment of the invention will comprise a heterologousnucleic acid sequence with adapted codon usage. Such a sequence encodesthe native amino acid sequence of the host from which it is derived, buthas a different nucleic acid sequence, i.e. a nucleic acid sequence inwhich certain codons have been replaced by other codons encoding thesame amino acid but which are more readily used by the host strain beingused for expression. This can lead to better expression of theheterologous nucleic acid sequence. This is common practice to a personskilled in the art. This adapted codon usage can be carried out on thebasis of known codon usage of fungal vis-a-vis non-fungal codon usage.It can also be even more specifically adapted to codon usage ofChrysosporium itself. The similarities are such that codon usage asobserved in Trichoderma, Humicola and Aspergillus should enable exchangeof sequences of such organisms without adaptation of codon usage.Details are available to the skilled person concerning the codon usageof these fungi and are incorporated herein by reference.

The invention is not restricted to the above mentioned recombinantChrysosporium strains, but also covers a recombinant Chrysosporiumstrain comprising a nucleic acid sequence encoding a homologous proteinfor a Chrysosporium strain, said nucleic acid sequence being operablylinked to an expression-regulating region and said recombinant strainexpressing more of said protein than the corresponding non-recombinantstrain under the same conditions. In the case of homologous polypeptideof interest such is preferably a neutral or alkaline enzyme like ahydrolase, a protease or a carbohydrate degrading enzyme as alreadydescribed elsewhere. The polypeptide may also be acidic. Preferably therecombinant strain will express the polypeptide in greater amounts thanthe non-recombinant strain. All comments mentioned vis-a-vis theheterologous polypeptide are also valid (mutatis mutandis) for thehomologous polypeptide cellulase.

Thus the invention also covers genetically engineered Chrysosporiumstrains wherein the sequence that is introduced can be of Chrysosporiumorigin. Such a strain can, however, be distinguished from nativelyoccurring strains by virtue of for example heterologous sequences beingpresent in the nucleic acid sequence used to transform or transfect theChrysosporium, by virtue of the fact that multiple copies of thesequence encoding the polypeptide of interest are present or by virtueof the fact that these are expressed in an amount exceeding that of thenon-engineered strain under identical conditions or by virtue of thefact that expression occurs under normally non-expressing conditions.The latter can be the case if an inducible promoter regulates thesequence of interest contrary to the non-recombinant situation or ifanother factor induces the expression than is the case in thenon-engineered strain. The invention as defined in the precedingembodiments is not intended to cover naturally occurring Chrysosporiumstrains. The invention is directed at strains derived throughengineering either using classical genetic technologies or geneticengineering methodologies.

All the recombinant strains of the invention can comprise a nucleic acidsequence encoding a heterologous protein selected fromcarbohydrate-degrading enzymes (cellulases, xylanases, mannanases,mannosidases, pectinases, amylases, e.g. glucoamylases,.alpha.-amylases, alpha- and beta-galactosidases, .alpha.- andβ-glucosidases, β-glucanases, chitinases, chitanases), proteases(endoproteases, amino-proteases, amino- and carboxy-peptidases,keratinases), other hydrolases (lipases, esterases, phytases),oxidoreductases (catalases, glucose-oxidases) and transferases(transglycosylases, transglutaminases, isomerases and invertases).

TABLE A pH range where enzymes retain activity and/or stability pH rangeretaining >50% pH range retaining >70% Stability enzymatic activityenzymatic activity (20 h, 50° C.) RBB- Other RBB- Other % from maxSample CMCase CMCase Substrates CMCase CMCase substrates pH 7.5/8 30 Kdprotease (alkaline) 30 kD — — 12.5  — — 12.0  — Xyl (alkaline) — — 10.0 — — 8.5 80 51 kD Xyl — — 8.0 — — 7.5 — 60 kD Xyl — — 9.5 — — 9.0 85 45kD endo 7.0 8.0 — 6.5 7.0 — 75 55 kD endo 8.0 8.0 — 7.0 7.0 — 55 25kD(21.8 kD′)endo 7.5 10.0  — 6.5 9.0 — 80 43 kD(39.6 kD*)endo 8.0 8.0 —7.2 7.2 — — 45 kD a,i-Gal/B-Glue — — 6.8 — — 5.7 — 48 kD CBH with13-Gluctraces 5.2 7.5 8.0 5.0 6.8 — — 55 kD CBH 8.0 9.0 — 7.4 8.5 — 70 65 kDPGU — — 8.0 — — 7.3 — 90 kD — — 9.0 — — 9.0 — protease 100 kD — — 9.0 —— 9.0 — esterase *molecular weights (by MALDI) Note: all other molecularweights by SDS PAGE enzymes were taken in equal protein contents * xyl =xylanase endo = endoglucanase gal = galactosidase gluc = glucosidase CBN= cellbiohydrolase PGU = polygalacturonase

TABLE B Activities of enzymes isolated from ultrafiltrate from 18-25strain toward different substrates (pH 5), units/mg protein RBB- CMC-CMC b-Gh1- CMC CMC 41 FP (vise) can pNP-a G pNP-a G Cellobiose Sample pI50° C. 40° C. 40° C. 50° C. 40° C. 50° C. 40° C. 40° C. 40° C. 30 kD 8.90 0 0 0 0 0 — 0 0 protease 30 kD Xyl 9.1 0.1 2 0.1 0.16 0.1 0 — 0 — 51kD Xyl 8.7 0.1 4.2 — 0.19 — 0 — 0 — 60 kD Xyl 4.7 0 — — 0 — 0 111 0 — 45kD endo 6 51 86 7.6 0.2 47 36 — 0 — 55 kD endo 4.9 47 94 7.7 0.3 39 25 —0 — 25 kD 4.1 19 15 3.9 0.3 11 3.8 — 0 0 (21.8 kD*) endo 43 kD 4.2 0.430.2 0.1 0 0.2 0.2 — 0 0 (39.6 kD*) endo 45 kD a, b 4.2 0 0 0 0 0.01 0.010 0.4 0.06 Gal/b-Ghuc 48 kD CDH 4.4 0.67 1.3 1.2 0.4 0.8 0.77 0 1.7 0.08with b- 0 Gluc traces + glcuono-d- lactone 55 kD CDH 4.4 0.7 0.16 0.270.4 0.1 0.1 — 0.05 0.08 with b- 0 Gluc traces + glcuono-00-d- lactone 65kD PGU 4.4 0 0 0 0 0 0 — 0 0 90 kD 4.2 — — — — — — — — — protease 100 kD4.5 0 0 0 0 0 0 0 0 0 esterase MUF- MUF Poly galacturonic AvicelMUF-cellobioside lactoside xyloside lactose Xylan acid Sample 40° C. 40°C. 40° C. 40° C. 50° C. 50° C. 50° C. 30 kD 0 0 0 0 0 0 0 protease 30 kDXyl 0 0 0 0 — 25 0 51 kD Xyl 0 0 0 0 — 19 0 60 kD Xyl 0 0.14 0.02 0.04 —16.3 0 45 kD endo 0.5 0 0 0 — 1 — 55 kD endo 0.3 0 0 0 — 0 — 25 kD 0.050 0 — 0 0.03 0 (21.8 kD*) endo 43 kD 0 0 0 — 0 0 0 (39.6 kD*) endo 45 kDa, b 0 0 0 — 0.01 0 0.1 Gal/b-Ghuc 48 kD CDH 0 0.2 0.36 — 0 0 0.1 withb- 0.36 Gluc traces + glcuono-d- lactone 55 kD CDH 0.46 0.2 0.7 — 0 0.10 with b- 0.14 0.6 Gluc traces + glcuono-00-d- lactone 65 kD PGU 0 0 0 —0 0 1 90 kD — — — — — — — protease 100 kD 0 0 0 0 0 0 0 esterase MUF-pNP-a NNP- Dyed pNP glucoside Galactomannan galactoside abgalactosidecasein″ butrate Sample 40° C. 50° C. 40° C. 40° C. 50° C. 60° C. 30 kD 00 0 0 0.4 0 protease 30 kD Xyl 0 0 0 — 0 0 51 kD Xyl 0 0 0 — 0 0 60 kDXyl 0 0 0 0 0 0 45 kD endo 0 12 0 — 0 0 55 kD endo 0 0.4 0 — 0 0 25 kD —0 0 0 0 0 (21.8 kD*) endo 43 kD — 0 0 0 0 0 (39.6 kD*) endo 45 kD a, b0.1 0.2 0.2 0.3 0 1.7 Gal/b-Ghuc 48 kD CDH 0.4 0 0 0 0 2.3 with b- Gluctraces + glcuono-d- lactone 55 kD CDH — 0 0 0 0 0 with b- 0.01 Gluctraces + glcuono-00-d- lactone 65 kD PGU — 0 0 0 0 0 90 kD — — — — 0.01— protease 100 kD 0 0 0 0 0 0.8 esterase *molecular weights, (by MALDI)**activity toward dyed casein was expressed in arbitrary units/mg

The most interesting products to be produced according to invention arecellulases, xylanases, pectinases, lipases and proteases, whereincellulases and xylanases cleave beta-1,4-bonds, and cellulases compriseendoglucanases, cellobiohydrolases and beta-glucosidases. These proteinsare extremely useful in various industrial processes known in the art.Specifically for cellulases we refer e.g. to WO 98/15633 describingcellobiohydrolases and endoglucanases of use. The contents of saidapplication are hereby incorporated by reference. We also refer toTables A and B providing further details of interesting Chrysosporiumproteins.

It was found according to the invention, that Chrysosporium mutants canbe made that have reduced expression of protease, thus making them evenmore suitable for the production of proteinaceous products, especiallyif the proteinaceous product is sensitive to protease activity. Thus theinvention also involves a mutant Chrysosporium strain which producesless protease than non-mutant Chrysosporium strain, for example lessthan C. lucknowense strain C1 (VKM F-3500 D). In particular the proteaseactivity of such strains is less than half the amount, more inparticular less than 30% of the amount produced by C1 strain. Thedecreased protease activity can be measured by known methods, such as bymeasuring the halo formed on skim milk plates or BSA degradation.

An embodiment of the invention that is of particular interest is arecombinant Chrysosporium according to the invention wherein the nucleicacid sequence encoding the polypeptide of interest encodes a polypeptidethat is inactivated or unstable at acid pH i.e. pH below 6, even belowpH 5.5, more suitably even below pH 5 and even as low as or lower thanpH 4. This is a particularly interesting embodiment, as the generallydisclosed fungal expression systems are not cultured under conditionsthat are neutral to alkaline, but are cultured at acidic pH. Thus thesystem according to the invention provides a safe fungal expressionsystem for proteins or polypeptides that are susceptible to beinginactivated or are unstable at acid pH.

Quite specifically a recombinant strain as defined in any of theembodiments according to the invention, wherein the nucleic acidsequence encoding the polypeptide of interest encodes a protein orpolypeptide exhibiting optimal activity and/or stability at a pH above5, preferably at neutral or alkaline pH (i.e. above 7) and/or at a pHhigher than 6, is considered a preferred embodiment of the invention.More than 50%, more than 70% and even more than 90% of optimalactivities at such pH values are anticipated as being particularlyuseful embodiments. A polypeptide expressed under the cultivationconditions does not necessarily have to be active at the cultivationconditions, in fact it can be advantageous for it to be cultured underconditions under which it is inactive as its active form could bedetrimental to the host. This is the case for proteases for example.What is however required is for the protein or polypeptide to be stableunder the cultivation conditions. The stability can be thermalstability. It can also be stability against specific compositions orchemicals, such as are present for example in compositions or processesof production or application of the polypeptide or protein of interest.Linear alkylbenzene sulfonate (LAS) in detergent compositions comprisingcellulases or lipases, etc. is an example of a chemical oftendetrimental to proteins. The time periods of use in applications canvary from short to long exposure so stability can be over a varyinglength of time varying per application. The skilled person will be ableto ascertain the correct conditions on a case by case basis. One can usea number of commercially available assays to determine the optimalactivities of the various enzymatic products. The catalogues of Sigmaand Megazyme for example show such. Specific examples of tests arementioned elsewhere in the description. The manufacturers provideguidance on the application.

We have surprisingly found that a Chrysosporium strain that can besuitably used to transform or transfect with the sequence of interest tobe expressed is a strain exhibiting relatively low biomass. We havefound that Chrysosporium strains having a biomass two to five timeslower than that of Trichoderma reesei when cultured to a viscosity of200-600 cP at the end of fermentation and exhibiting a biomass of 10 to20 times lower than that of Aspergillus niger when cultured to aviscosity of 1500-2000 cP under corresponding conditions, i.e. theirrespective optimal cultivation conditions can provide a high level ofexpression. This level of expression far exceeds that of the twocommercial reference strains at a much lower biomass and at much lowerviscosity. This means that the yield of expression of such Chrysosporiumstrains will be appreciably higher than from Aspergillus niger andTrichoderma reesei. Such a transformed or transfected Chrysosporiumstrain forms a suitable embodiment of the invention.

We find a biomass of 0.5-1.0 g/l for Chrysosporium strain C1 (18-25) asopposed to 2.5-5.5 g/l for Trichoderma reesei and 5-10 g/l ofAspergillus niger under the above described conditions. In the Exampleswe provide details of this process.

In a suitable embodiment a recombinant Chrysosporium strain according tothe invention produces protein or polypeptide in at least the amountequivalent to the production in moles per liter of cellulase by thestrain UV13-6 or NG7C-19, and most preferably at least equivalent to orhigher than that of the strain UV 18-25 under the corresponding oridentical conditions, i.e. their respective optimal cultivationconditions.

Unexpectedly we have also found that expression and secretion rates areexceedingly high when using a Chrysosporium strain exhibiting themycelial morphology of strain UV18-25 i.e. fragmented short mycelia.Thus a recombinant strain according to the invention will preferablyexhibit such morphology. The invention however also coversnon-recombinant strains or otherwise engineered strains of Chrysosporiumexhibiting this novel and inventive characteristic. Also covered by theinvention is a recombinant Chrysosporium strain in any of theembodiments described according to the invention further exhibitingreduced sporulation in comparison to C1, preferably below that of strainUV13-6, preferably below that of NG7C-19, preferably below that ofUV18-25 under equivalent fermenter conditions. Also covered by theinvention is a recombinant Chrysosporium strain in any of theembodiments described according to the invention further exhibiting atleast the amount of protein production ratio to biomass in comparison toC1, preferably in comparison to that of any of strains UV13-6, NG7C-19and UV18-25 under equivalent fermenter conditions. The invention howeveralso covers non-recombinant strains or otherwise engineered strains ofChrysosporium exhibiting this novel and inventive characteristic as suchor in combination with any of the other embodiments.

Another attractive embodiment of the invention also covers a recombinantChrysosporium strain exhibiting a viscosity below that of strainNG7C-19, preferably below that of UV18-25 under corresponding oridentical fermenter conditions. The invention however also coversnon-recombinant strains or otherwise engineered strains of Chrysosporiumexhibiting this novel and inventive characteristic as such or incombination with any of the other embodiments. We have determined thatthe viscosity of a culture of UV 18-25 is below 10 cP opposed to that ofTrichoderma reesei being of the order 200-600 cP, with that ofAspergillus niger being of the order 1500-2000 cP under their respectiveoptimal culture conditions at the end of fermentation. The process usedfor such determination is provided in the examples.

Viscosity can be assessed in many cases by visual monitoring. Thefluidity of the substance can vary to such a large extent that it can benearly solid, sauce like or liquid. Viscosity can also readily beascertained by Brookfield rotational viscometry, use of kinematicviscosity tubes, falling ball viscometer or cup type viscometer. Theyields from such a low viscosity culture are higher than from thecommercial known higher viscosity cultures per time unit and per cell.

The processing of such low viscosity cultures according to the inventionis advantageous in particular when the cultures are scaled up. Thesubject Chrysosporium strains with the low viscosity perform very wellin cultures as large as up to 150,000 liter cultures. Thus any culturesize up to 150,000 liters provides a useful embodiment of the invention.Any other conventional size of fermentation should be carried out wellwith the strains according to the invention. The reasoning behind thisis that problems can arise in large scale production with the formationof aggregates that have mycelia that are too dense and/or are unevenlydistributed. The media as a result cannot be effectively utilised duringthe culture thus leading to an inefficient production process inparticular in large scale fermentations i.e. over 150,000 liters.Aeration and mixing become problematic leading to oxygen and nutrientstarvation and thus reduced concentration of productive biomass andreduced yield of polypeptide during the culture and/or can result inlonger fermentation times. In addition high viscosity and high shear arenot desirable in commercial fermentation processes and in currentcommercial processes they are the production limiting factors. All thesenegative aspects can be overcome by the Chrysosporium host according tothe invention which exhibits much better characteristics thanTrichoderma reesei, Aspergillus niger and Aspergillus oryzae that arecommercially used in this respect i.e. exhibits better proteinproduction levels and viscosity properties and biomass figures.

A Chrysosporium strain selected from C1, UV13-6, NG7C-19 and UV18-25illustrates various aspects of the invention exceedingly well. Theinvention however also covers recombinant strains or otherwiseengineered strains of Chrysosporium derived from the four depositedstrains that also exhibit any of the novel and inventive characteristicsas such or in combination. The deposit data for these strains have beenpresented elsewhere in the description. The invention also coversrecombinant strains or otherwise engineered strains of Chrysosporiumderived from the four deposited strains that also exhibit any of thenovel and inventive characteristics as such or in combination. AChrysosporium strain according to the invention also comprises a strainexhibiting under the corresponding culture conditions a biomass at leasttwice as low as that of Trichoderma reesei, suitably even more up to 5times lower than that of Trichoderma reesei, specifically of aTrichoderma reesei exhibiting a viscosity of 200-600 cP as disclosedunder the conditions of the examples. A Chrysosporium strain accordingto the invention also comprises a strain producing the polypeptide in atleast the amount in moles per liter of cellulase by the strain C1,UV13-6, NG7C-19 or UV18-25 under the corresponding or identicalconditions.

Chrysosporium strains according to the invention are further preferredif they exhibit optimal growth conditions at neutral to alkaline pH andtemperatures of 25-43° C. A preference can exist for neutral and evenfor alkaline pH. Such production conditions are advantageous to a numberof polypeptides and proteins, in particular those susceptible to attackby acidic pH or those that are inactive or unstable at low temperatures.It is however also an embodiment of the invention to includeChrysosporium strains that can be cultured at acidic pH as this can beuseful for certain proteins and polypeptides. A suitable acidic pH liesfrom 7.0. An acidic pH lower than 6.5 is envisaged as providing a goodembodiment of the invention. A pH around 5.0-7.0 is also a suitableembodiment. A neutral pH can be 7.0 or around 7 e.g. 6.5-7.5. As statedelsewhere the pH of optimal interest depends on a number of factors thatwill be apparent to the person skilled in the art. A pH higher than 7.5is alkaline, suitably between 7.5-9.0 can be used.

When comparing data of strains according to the invention with otherstrains perhaps having other optimal conditions (e.g. Aspergillus andTrichoderma) for viscosity measurements, biomass determination orprotein production comparisons should be made using the relevant optimalconditions for the relevant strain. This will be obvious to the personskilled in the art.

A Chrysosporium strain according to any of the above-mentionedembodiments of the invention, said strain further exhibiting productionof one or more of the fungal enzymes selected from thecarbohydrate-degrading enzymes, proteases, other hydrolases,oxidoreductase, and transferases mentioned above is considered aparticularly useful embodiment of the invention. The most interestingproducts are specifically cellulases, xylanases, pectinases, lipases andproteases. Also useful as embodiment of the invention however is aChrysosporium strain exhibiting production of one or more fungal enzymesthat exhibit neutral or alkaline optimal stability and/or activity,preferably alkaline optimal stability and/or activity, said enzyme beingselected from carbohydrate-degrading enzymes, hydrolases and proteases,preferably hydrolases and carbohydrate-degrading enzymes. In the case ofnon-recombinant Chrysosporium, such enzymes are suitably other thancellulase as disclosed in WO 98/15633. Enzymes of particular interestare xylanases, proteases, esterases, alpha-galactosidases,beta-galactosidases, beta-glucanases and pectinases. The enzymes are notlimited to the aforementioned. The comments vis-a-vis stability andactivity elsewhere in the description are valid here also.

The invention also covers a method of producing a polypeptide ofinterest, said method comprising culturing a Chrysosporium strain in anyof the embodiments according to the invention under conditionspermitting expression and preferably secretion of the polypeptide andrecovering the subsequently produced polypeptide of interest.

Where protein or polypeptide is mentioned, variants and mutants e.g.substitution, insertion or deletion mutants of naturally occurringproteins are intended to be included that exhibit the activity of thenon-mutant. The same is valid vis-a-vis the corresponding nucleic acidsequences. Processes such as gene shuffling, protein engineering anddirected evolution, site directed mutagenesis and random mutagenesis areprocesses through which such polypeptides, variants or mutants can beobtained. U.S. Pat. No. 5,223,409, U.S. Pat. No. 5,780,279 and U.S. Pat.No. 5,770,356 provide teaching of directed evolution. Using this processa library of randomly mutated gene sequences created for example by geneshuffling via error prone PCR occurs in any cell type. Each gene has asecretion region and an immobilising region attached to it such that theresulting protein is secreted and stays fixed to the host surface.Subsequently conditions are created that necessitate the biologicalactivity of the particular polypeptide. This occurs for a number ofcycles ultimately leading to a final gene with the desiredcharacteristics. In other words a speeded up directed process ofevolution. U.S. Pat. No. 5,763,192 also describes a process forobtaining DNA, RNA, peptides, polypeptides or protein by way ofsynthetic polynucleotide coupling stochastically generated sequences,introduction thereof into a host followed by selection of the host cellwith the corresponding predetermined characteristic.

Another application of the method of the present invention is in theprocess of “directed evolution”, wherein novel protein-encoding DNAsequences are generated, the encoded proteins are expressed in a hostcell, and those sequences encoding proteins having a desiredcharacteristic are mutated and expressed again. The process is repeatedfor a number of cycles until a protein with the desired characteristicsis obtained. Gene shuffling, protein engineering, error-prone PCR,site-directed mutagenesis, and combinatorial and random mutagenesis areexamples of processes through which novel DNA sequences encodingexogenous proteins can be generated. U.S. Pat. Nos. 5,223,409, 5,780,279and 5,770,356 provide teaching of directed evolution. See also Kuchnerand Arnold, Trends in Biotechnology, 15:523-530 (1997); Schmidt-Dannertand Arnold, Trends in Biotech., 17:135-136 (1999); Arnold and Volkov,Curr. Opin. Chem. Biol., 3:54-59 (1999); Zhao et al., Manual ofIndustrial Microbiology and Biotechnology, 2nd Ed., (Demain and Davies,eds.) pp. 597-604, ASM Press, Washington D.C., 1999; Arnold andWintrode, Encyclopedia of Bioprocess Technology: Fermentation,Biocatalysis, and Bioseparation, (Flickinger and Drew, eds.) pp.971-987, John Wiley & Sons, New York, 1999; and Minshull and Stemmer,Curr. Opin. Chem. Biol. 3:284-290.

An application of combinatorial mutagenesis is disclosed in Hu et al.,Biochemistry. 1998 37:10006-10015. U.S. Pat. No. 5,763,192 describes aprocess for obtaining novel protein-encoding DNA sequences bystochastically generating synthetic sequences, introducing them into ahost, and selecting host cells with the desired characteristic. Methodsfor effecting artificial gene recombination (DNA shuffling) includerandom priming recombination (Z. Shao, et al., Nucleic Acids Res.,26:681-683 (1998)), the staggered extension process (H. Zhao et al.,Nature Biotech., 16:258-262 (1998)), and heteroduplex recombination (A.Volkov et al., Nucleic Acids Res., 27:e18 (1999)). Error-prone PCR isyet another approach (Song and Rhee, Appl. Environ. Microbiol.66:890-894 (2000)).

There are two widely-practiced methods of carrying out the selectionstep in a directed evolution process. In one method, the proteinactivity of interest is somehow made essential to the survival of thehost cells. For example, if the activity desired is a cellulase activeat pH 8, a cellulase gene could be mutated and introduced into the hostcells. The transformants are grown with cellulose as the sole carbonsource, and the pH raised gradually until only a few survivors remain.The mutated cellulase gene from the survivors, which presumably encodesa cellulase active at relatively high pH, is subjected to another roundof mutation, and the process is repeated until transformants that cangrow on cellulose at pH 8 are obtained. Thermostable variants of enzymescan likewise be evolved, by cycles of gene mutation and high-temperatureculturing of host cells (Liao et al., Proc. Natl. Acad. Sci. USA83:576-580 (1986); Giver et al., Proc. Natl. Acad. Sci. USA.95:12809-12813 (1998).

An alternative to the massively parallel “survival of the fittest”approach is serial screening. In this approach, individual transformantsare screened by traditional methods, such as observation of cleared orcolored zones around colonies growing on indicator media, calorimetricor fluorometric enzyme assays, immunoassays, binding assays, etc. Seefor example Joo et al., Nature 399:670-673 (1999), where a cytochromeP450 monooxygenase not requiring NADH as a cofactor was evolved bycycles of mutation and screening; May et al., Nature Biotech. 18:317-320(2000), where a hydantoinase of reversed stereoselectivity was evolvedin a similar fashion; and Miyazaki et al., J. Mol. Biol. 297:1015-1026(2000), where a thermostable subtilisin was evolved.

Standard cloning and protein or polypeptide isolation techniques can beused to arrive at the required sequence information. Parts of knownsequences can be used as probes to isolate other homologues in othergenera and strains. The nucleic acid sequence encoding a particularenzyme activity can be used to screen a Chrysosporium library forexample. A person skilled in the art will realise which hybridisationconditions are appropriate. Conventional methods for nucleic acidhybridisation construction of libraries and cloning techniques aredescribed in Sambrook et al (Eds) (1989) In “Molecular Cloning. ALaboratory Manual” Cold Spring Harbor Press, Plainview, N.Y., andAusubel et al (Eds) “Current Protocols in Molecular Biology” (1987) JohnWiley and Sons, New York. The relevant information can also be derivedfrom later handbooks and patents, as well as from various commerciallyavailable kits in the field.

In an alternative embodiment, said method comprises culturing a strainaccording to the invention under conditions permitting expression andpreferably secretion of the protein or polypeptide or precursor thereofand recovering the subsequently produced polypeptide and optionallysubjecting the precursor to additional isolation and purification stepsto obtain the polypeptide of interest. Such a method may suitablycomprise a cleavage step of the precursor into the polypeptide orprecursor of interest. The cleavage step can be cleavage with a Kex-2like protease, any basic amino acid paired protease or Kex-2 for examplewhen a protease cleavage site links a well secreted protein carrier andthe polypeptide of interest. A person skilled in the art can readilyfind Kex-2-like protease sequences as consensus sequence details forsuch are available and a number of alternatives have already beendisclosed e.g. furin.

Suitably in a method for production of the polypeptide according to anyof the embodiments of the invention the cultivation occurs at pH higherthan 5, preferably 5-10, more preferably 6-9. Suitably in such a methodthe cultivation occurs at a temperature between 25-43° C., preferably30-40° C. The Chrysosporium strain used in the method according to theinvention is quite suitably a recombinant Chrysosporium strain accordingto any of the embodiments disclosed. The method according to theinvention in such a case can further be preceded by the step ofproduction of a recombinant Chrysosporium strain according to theinvention. The selection of the appropriate conditions will depend onthe nature of the polypeptide to be expressed and such selection lieswell within the realm of normal activity of a person skilled in the art.

The method of production of a recombinant Chrysosporium strain accordingto the invention is also part of the subject invention. The methodcomprises stably introducing a nucleic acid sequence encoding aheterologous or homologous polypeptide into a Chrysosporium strain, saidnucleic acid sequence being operably linked to an expression regulatingregion, said introduction occurring in a manner known per se fortransforming filamentous fungi. As stated above numerous referenceshereof are available and a small selection has been cited. Theinformation provided is sufficient to enable the skilled person to carryout the method without undue burden. The method comprises introductionof a nucleic acid sequence comprising any of the nucleic acid elementsdescribed in the various embodiments of the recombinant Chrysosporiumaccording to the invention as such or in combination.

By way of example the introduction can occur using the protoplasttransformation method. The method is described in the examples.Alternative protoplast or spheroplast transformation methods are knownand can be used as have been described in the prior art for otherfilamentous fungi. Details of such methods can be found in many of thecited references and are thus incorporated by reference. A methodaccording to the invention suitably comprises using a non-recombinantstrain of Chrysosporium according to the invention as starting materialfor introduction of the desired sequence encoding the polypeptide ofinterest.

The subject invention also covers a method of producing Chrysosporiumenzyme, said method comprising culturing a Chrysosporium strainaccording to any of the embodiments of the invention as described abovein or on a cultivation medium at pH higher than 5, preferably 5-10, morepreferably 6-9, suitably 6-7.5, 7.5-9 as examples of neutral andalkaline pH ranges.

The subject invention also covers such a method using a cultivationmedium at a temperature between 25-43° C., preferably 30-40° C. Thecombination of preferred pH and temperature is an especially preferredembodiment of the method of producing Chrysosporium enzyme according tothe invention.

More in general the invention further covers a method of producingenzymes exhibiting neutral or alkaline optimal activity and/orstability, preferably alkaline optimal activity and/or stability. Thepreferred ranges vis-a-vis pH and optimal activity as well as assayswith which to determine such have been provided elsewhere in thedescription. The enzyme should be selected from carbohydrate-degradingenzymes, proteases, other hydrolases, oxidoreductases, and transferases,as described above, said method comprising cultivating a host celltransformed or transfected with the corresponding enzyme-encodingnucleic acid sequence. Suitably such an enzyme will be a Chrysosporiumenzyme. A suitable method such as this comprises production specificallyof cellulase, xylanase, pectinase, lipase and protease, whereincellulase and xylanase cleave β-1,4-bonds and cellulase comprisesendoglucanase, cellobiohydrolase and β-glucosidase. The method accordingto the invention can comprise cultivating any Chrysosporium hostaccording to the invention comprising nucleic acid encoding suchaforementioned enzymes. Suitably the production of non-recombinantChrysosporium hosts according to the invention is directed at productionof carbohydrate degrading enzymes, hydrolases and proteases. In such acase the enzyme is suitably other than a cellulase. Suitable examples ofproducts to be produced are given in Tables A and B. Methods ofisolating are analogous to those described in WO 98/15633 and areincorporated by reference.

The enzymes produced by Chrysosporium strains according to the inventionare also covered by the invention. Enzymes of Chrysosporium origin ascan be isolated from non-recombinant Chrysosporium strains according tothe invention are also covered. They exhibit the aforementionedstability, activity characteristics. Suitably they are stable in thepresence of LAS. In particular proteases with pI 4-9.5, proteases with aMW of 25-95 kD, xylanases with pI between 4.0 and 9.5, xylanases with MWbetween 25 and 65 kD, endoglucanases with a pI between 3.5 and 6.5,endoglucanases with MW of 25-55 kDa, β-glucosidases, .alpha.,β-galactosidases with a pI of 4-4.5, β-glucosidases, a,β-galactosidaseswith a MW of 45-50 kDa, cellobiohydrolases of p14-5, cellobiohydrolasesof MW 45-75 kDa, e.g. a MW of 55 kD and pI 4.4, polygalacturonases, witha pI of 4.0-5.0 polygalacturonase of 60-70 kDa, e.g. 65 kDa, esteraseswith a pl 4-5, and esterases with a MW of 95-105 kDa with theafore-mentioned stability, activity characteristics are claimed. Themolecular weights (MW) are those determined by SDS-PAGE. Thenon-recombinant i.e. natively occurring enzyme is other than cellulaseas disclosed in WO 98/15633. An enzyme as disclosed in WO 98/15633 isexcluded. Enzymes according to the invention are represented by theenzymes of Table B. Enzymes with combinations of the pI values andmolecular weights mentioned above are also covered.

The invention is also concerned with the (over)production of non-proteinproducts by the mutant (recombinant) strains of the invention. Suchnon-protein products include primary metabolites such as organic acids,amino acids, and secondary metabolites such as antibiotics, e.g.penicillins and cephalosporins, and other therapeutics. These productsare the result of combinations of biochemical pathways, involvingseveral fungal genes of interest. Fungal primary and secondarymetabolites and procedures for producing these metabolites in fungalorganisms are well known in the art. Examples of the production ofprimary metabolites have been described by Mattey M., The Production ofOrganic Acids, Current Reviews in Biotechnology, 12, 87-132 (1992).Examples of the production of secondary metabolites have been describedby Penalva et al. The Optimization of Penicillin Biosynthesis in Fungi,Trends in Biotechnology 16, 483-489 (1998).

EXAMPLES Examples of Biomass and Viscosity Determinations

The following operating parameter data ranges have been determined forfungal fermentations using three different fungal organisms. The threefungal organisms compared are: Trichoderma longibrachiatum (formerly T.reesei), Aspergillus niger and Chrysosporium lucknowense (UV 18-25).

Viscosity:

Viscosity is determined on a Brookfield LVF viscometer using the smallsample adapter and spindle number 31.

Turn the water-circulating pump on 5 minutes prior to viscometer use toequilibrate the water jacket. The water bath temperature should be 30°C.

Obtain a fresh sample of fermentation broth and place 10 ml of the brothin the small sample spindle. Select the spindle speed to give a readingin the range 10-80. Wait four (4) minutes and take the reading from theviscometer scale. Multiply the reading by the factor given below to getthe viscosity in centipoise (cP).

Spindle Speed Multiplication Factor 6 50 12 25 30 10 60 5

The following viscosity ranges have been determined for fermentationsusing the specified fungal organism using the above procedure:

Viscosity in cP T. longibrachiatum 200-600 A. niger 1,500-2,000 C.lucknowense (UV18-25) LT 10

Biomass:

Biomass is determined by the following procedure:

Preweigh 5.5 cm filter paper (Whatman 54) in an aluminium weighing dish.Filter 5.0 ml whole broth through the 5.5 cm paper on a Buchner funnel,wash the filter cake with 10 ml deionised water, place the washed cakeand filter in a weighing pan and dry overnight at 60° C. Finish dryingat 100° C. for 1 hour, then place in desiccator to cool.

Measure the weight of dried material. Total biomass (g/l) is equal tothe difference between the initial and finals weights multiplied by 200.The following biomass ranges have been determined for fermentationsusing the specified fungal organism using the above procedure:

Biomass in g/l T. longibrachiatum 2.5-5 A. niger   5-10 C. lucknowense(UV18-25) 0.5-1

Protein:

Protein levels were determined using the BioRad Bradford Assay Procedurefrom BioRad Laboratories. Protein levels were highest for theChrysosporium.

The data presented above represent values determined 48 hours into thefermentation process until fermentation end; All values of Aspergilliand Trichoderma are for commercially relevant fungal organisms andreflect actual commercial data.

A fungal strain such as C. lucknowense (UV18-25) has the advantage thatthe low viscosity permits the use of lower power input and/or shear inthe fermentation to meet oxygen demands for those cases where shearstress on the product may be detrimental to productivity due to physicaldamage of the product molecule. The lower biomass production at highprotein production indicates a more efficient organism in the conversionof fermentation media to product. Thus the Chrysosporium provides betterbiomass and viscosity data whilst also delivering at least as muchprotein, and in fact a lot more protein than the two commercially usedsystems which obviously are better than for typically depositedAspergillus or Trichoderma reesei strains in general public collections.

The high protein production with low biomass concentration produced byC. lucknowense (UV18-25) would allow development of fermentationconditions with higher multiples of increase in biomass, if increasingbiomass results in increased productivity, for the desired productbefore reaching limiting fermentation conditions. The present highlevels of biomass and viscosity produced by the T. longibrachiatum andA. niger organisms restrict the increase of biomass as the presentlevels of biomass and viscosity are near limiting practical fermentationconditions.

Examples of Transformation Comparing Chrysosporium, Trichoderma andTolypocladium Geodes

Two untransformed Chrysosporium C1 strains and one Trichoderma reeseireference strain were tested on two media (Gs pH 6.8 and Pridham agar,PA, pH 6,8). To test the antibiotic resistance level spores werecollected from 7 day old PDA plates. Selective plates were incubated at32° C. and scored after 2, 4, and 5 days. It followed that the C-1strains NG7C-19 and UV18-25 clearly have a low basal resistance levelboth to phleomycin and hygromycin. This level is comparable to that fora reference T. reesei commonly used laboratory strain. Thus there isclear indication these two standard fungal selectable markers can beused well in Chrysosporium strains. Problems with other standard fungalselectable markers should not be expected.

Selection of Sh-ble (phleomycin-resistance) transformed Chrysosporiumstrains was successfully carried out at 50 μg/ml. This was also theselection level used for T. reesei thus showing that differentialselection can be easily achieved in Chrysosporium. The same comments arevalid for transformed strains with hygromycin resistance at a level of150 μg/ml.

TABLE C Gs (pH 6.8) Pridham Agar (PA, pH 6.8) NG7C-19 UV18-25 T.r.11D5NG7C-19 UV18-25 T.r.11D5 Phleomycin   7.5 Kg/m1 10 μg/ml 5-7.5 pg/ml 2.5μg/ml 10 Kg/m1 2.5 Kg/m1 Hygromycin 7.5-10 Kg/m1 10 μg/ml    10 lag/m1 15 Kg/m1 25 Kg/m1  15 pg/m1

The protoplast transformation technique was used on Chrysosporium basedon the most generally applied fungal transformation technology. Allspores from one 90 mm PDA plate were recovered in 8 ml IC1 andtransferred into a shake flask of 50 ml IC1 medium for incubation for 15hours at 35° C. and 200 rpm. After this the culture was centrifuged, thepellet was washed in MnP, brought back into solution in 10 ml MnP and 10mg/ml Caylase C.sub.3 and incubated for 30 minutes at 35° C. withagitation (150 rpm).

The solution was filtered and the filtrate was subjected tocentrifugation for 10 minutes at 3500 rpm. The pellet was washed with 10ml MnPCa.sup.2+. This was centrifuged for 10 minutes at 25° C. Then 50microliters of cold MPC was added. The mixture was kept on ice for 30minutes whereupon 2.5 ml PMC was added. After 15 minutes at roomtemperature 500 microliters of the treated protoplasts were mixed to 3ml of MnR Soft and immediately plated out on a MnR plate containingphleomycin or hygromycin as selection agent. After incubation for fivedays at 30° C. transformants were analysed (clones become visible after48 hours). Transformation efficiency was determined using 10microgrammes of reference plasmid pAN8-1¹⁹. The results are presented inthe following Table D.

TABLE D Transformation efficiency (using 10 lig of reference plasmidpAN8-1) T. reesei NG7C-19 UV18-25 Viability 106/200 p1 5 × 106/2001.11 5× 106/200 pi Transformants 2500  104  104 Per 200 [11 Transformants per106 2500 2000 2000 viable cells

The results show that the Chrysosporium transformants viability issuperior to that of Trichoderma. The transformability of the strains iscomparable and thus the number of transformants obtained in oneexperiment lies 4 times higher for Chrysosporium than for T. reesei.Thus the Chrysosporium transformation system not only equals thecommonly used T. reesei system, but even outperforms it. Thisimprovement can prove especially useful for vectors that are lesstransformation efficient than pAN8-1. Examples of such less efficienttransformation vectors are protein carrier vectors for production ofnon-fungal proteins which generally yield 10 times fewer transformants.

A number of other transformation and expression vectors were constructedwith homologous Chrysosporium protein encoding sequences and also withheterologous protein encoding sequences for use in transformationexperiments with Chrysosporium. The vector maps are provided in theFIGS. 6-11.

The homologous protein to be expressed was selected from the group ofcellulases produced by Chrysosporium and consisted of endoglucanase 6which belongs to family 6 (MW 43 kDa) and the heterologous protein wasendoglucanase 3 which belongs to family 12 (MW 25 kDa) of Penicillium.

pF6g comprises Chrysosporium endoglucanase 6 promoter fragment linked toendoglucanase 6 signal sequence in frame with the endoglucanase 6 openreading frame followed by the endoglucanase 6 terminator sequence.Transformant selection is carried out by using cotransformation with aselectable vector.

pUT1150 comprises Trichoderma reesei cellobiohydrolase promoter linkedto endoglucanase 6 signal sequence in frame with the endoglucanase 6open reading frame followed by the T. reesei cellobiohydrolaseterminator sequence. In addition this vector carries a second expressioncassette with a selection marker i.e. the phleomycin resistance gene(Sh-ble gene).

pUT1152 comprises Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to endoglucanase 6 signal sequence inframe with the endoglucanase 6 open reading frame followed by the A.nidulans anthranilate synthase (trpC) terminator sequence. In additionthis vector carries a second expression cassette with a selection markeri.e. the phleomycin resistance gene (Sh-ble gene).

pUT1155 comprises A. nidulans glyceraldehyde-3-phosphate dehydrogenase Apromoter linked to Trichoderma reesei cellobiohydrolase signal sequencein frame with the carrier protein Sh-ble which in turn is linked inframe to the endoglucanase 6 open reading frame followed by the A.nidulans trpC terminator sequence. This vector uses the technology ofthe carrier protein fused to the protein of interest which is known tovery much improve the secretion of the protein of interest.

pUT1160 comprises Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to Trichoderma reesei cellobiohydrolasesignal sequence in frame with the carrier protein Sh-ble which in turnis linked in frame to the endoglucanase 3 open reading frame ofPenicillium followed by the A. nidulans trpC terminator sequence.

pUT1162 comprises Trichoderma reesei cellobiohydrolase promoter linkedto endoglucanase 3 signal sequence in frame with the endoglucanase 3open reading frame of Penicillium followed by the T. reeseicellobiohydrolase terminator sequence. In addition this vector carries asecond expression cassette with a selection marker i.e. the phleomycinresistance gene (Sh-ble gene).

Further examples of expression systems include a Chrysosporiumendoglucanase 3 promoter fragment linked to endoglucanase 3 signalsequence in frame with the endoglucanase 3 open reading frame followedby the endoglucanase 3 terminator sequence. Transformant selection iscarried out by using cotransformation with a selectable vector.

Another example is a Chrysosporium lucknowense cellobiohydrolasepromoter linked to Penicillium endoglucanase 3 signal sequence in framewith the Penicillium endoglucanase 3 open reading frame followed by theChrysosporium cellobiohydrolase terminator sequence. In addition thisvector carries a second expression cassette with a selection marker i.e.the aceetamidase S gene (AmdS gene).

A further example comprises Chrysosporium glyceraldehyde-3-phosphatedehydrogenase 1 promoter linked to the Aspergillus niger glucoamylasesignal sequence and the glucoamylase open reading frame fused to thehuman Interleukine 6 open reading frame. In addition this vector carriesa second expression cassette with a selection marker i.e. the AmdS gene.

A still further example is a Aspergillus nidulansglyceraldehyde-3-phosphate dehydrogenase A promoter linked to theendoglucanase 5 open reading frame followed by a Aspergillus nidulansterminator sequence.

TABLE E Comparative transformations Tested in liquid Vector StrainTransformation No of transf. culture pUT1150 UV18-25 selection phleo 2855 T geodes selection phleo 144 5 pUT1152 UV18-25 cotransformation pAN8.1398 5 T geodes cotransformation pAN8.1 45 4 pF6g UV18-25cotransformation pAN8.1 252 6 T geodes cotransformation pAN8.1 127 5pUT1162 UV18-25 selection phleo >400 T geodes Not done yet

Table E shows the results of transformation of both ChrysosporiumUV18-25 and Tolypocladium geodes. The transformation protocol used isdescribed in the section for heterologous transformation.

Examples of Heterologous and Homologous Expression of ChrysosporiumTransformants

C1 strains (NG7C-19 and/or UV18-25) have been tested for their abilityto secrete various heterologous proteins: a bacterial protein(Streptoalloteichus hindustanus phleomycin-resistance protein, Sh ble),a fungal protein (Trichoderma reesei xylanase II, XYN2) and a humanprotein (the human lysozyme, HLZ).

The details of the process are as follows:

[1] C1 Secretion of Streptoalloteichus hindustanus Phleomycin-ResistanceProtein (Sh ble).

C1 strains NG7C-19 and UV18-25 have been transformed by the plasmidpUT720¹. This vector presents the following fungal expression cassette:

-   -   Aspergillus nidulans_glyceraldehyde-3-phosphate dehydrogenase        (gpdA) promoter²    -   A synthetic Trichoderma reesei cellobiohydrolase I (cbh1) signal        sequence^(1,3)    -   Streptoalloteichus hindustanus phleomycin-resistance gene Sh        ble⁴    -   Aspergillus nidulans tryptophan-synthase (trpC) terminator⁵

The vector also carries the beta-lactamase gene (bla) and E. colireplication origin from plasmid pUC18⁶. The detailed plasmid map isprovided in FIG. 2.

C1 protoplasts were transformed according to Durand et al.⁷ adapted toC1 (media & solutions composition is given elsewhere): All spores fromone 90 mm PDA plate of untransformed C1 strain were recovered in 8 mlIC1 and transferred into a shake flask with 50 ml IC1 medium forincubation 15 hours at 35° C. and 150 rpm. Thereupon, the culture wasspun down, the pellet washed in MnP, resolved in 10 ml MnP+10 mg/mlCaylase C.sub.3, and incubated 30 min at 35° C. with agitation (150rpm). The solution was filtered and the filtrate was centrifuged 10 minat 3500 rpm. The pellet was washed with 10 ml MnPCa²⁺. This was spundown 10 min at 3500 rpm and the pellet was taken up into 1 ml MnPCa²⁺.10 μg of pUT720 DNA were added to 200 μA of protoplast solution andincubated 10 min at room temperature (^(˜)20° C.). Then, 50 μl of coldMPC was added. The mixture was kept on ice for 30 min whereupon 2.5 mlPMC was added. After 15 min at room temperature 500 μl of the treatedprotoplasts were mixed to 3 ml of MnR Soft and immediately plated out ona MnR plate containing phleomycin (50 μg/ml at pH6.5) as selectionagent. After 5 days incubation at 30° C., transformants were analysed(clones start to be visible after 48 hours).

The Sh ble production of C1 transformants (phleomycin-resistant clones)was analysed as follows: Primary transformants were toothpicked toGS+phleomycin (5 μg/ml) plates and grown for 5 days at 32° C. forresistance verification. Each validated resistant clone was subclonedonto GS plates. Two subclones per transformant were used to inoculatePDA plates in order to get spores for liquid culture initiation. Theliquid cultures in IC1 were grown 5 days at 27° C. (shaking 200 rpm).Then, the cultures were centrifuged (5000 g, 10 min.) and 500 μl ofsupernatant were collected. From these samples, the proteins wereprecipitated with TCA and resuspended in Western Sample Buffer to 4mg/ml of total proteins (Lowry Method.sup.8). 10 g1 (about 40 μg oftotal proteins) were loaded on a 12% acrylamide/SDS gel and run (BioRadMini Trans-Blot system). Western blotting was conducted according toBioRad instructions (Schleicher & Schull 0.2 μm membrane) using rabbitanti-Sh ble antiserum (Cayla Cat. Ref #ANTI-0010) as primary antibody.

The results are shown in FIG. 1 and Table F:

TABLE F Sh ble estimated production levels in Cl Estimated Estimated Shble Sh ble quantity concentration in on the Western blot the productionmedia Untransformed NG7C-19 Not detectable NG7C-19::720 clone 4-1  25 ng0.25 mg/1 NG7C-19::720 clone 5-1  25 ng 0.25 mg/1 NG7C-19::720 clone 2-2250 ng  2.5 mg/1 Untransformed UV18-25 Not detectable UV18-25::720 clone1-2 500 ng   5 mg/1 UV18-25::720 clone 3-1 250 ng  2.5 mg/1

These data show that:

1) The heterologous transcription/translation signals from pUT720 arefunctional in Chrysosporium.2) The heterologous signal sequence of pUT720 is functional inChrysosporium.

3) Chrysosporium can be used a host for the secretion of an heterologousbacterial protein.

[2] C1 Secretion of the Human Lysozyme (HLZ).

C1 strains NG7C-19 and UV18-25 have been transformed by the plasmidpUT970G⁹. This vector presents the following fungal expression cassette:

-   -   Aspergillus nidulans_glyceraldehyde-3-phosphate dehydrogenase        (gpdA) promoter²    -   A synthetic Trichoderma _(—) reesei cellobiohydrolase I (cbh1)        signal sequence^(1,3)    -   Streptoalloteichus hindustanus phleomycin-resistance gene Sh        ble⁴ used as carrier-protein¹⁰    -   Aspergillus niger glucoamylase (glaA2) hinge domain cloned from        plasmid pAN56-2^(11,12)    -   A linker peptide (LGERK) featuring a KEX2-like protease cleavage        site¹    -   A synthetic human lysozyme gene (hlz)¹⁰    -   Aspergillus nidulans tryptophan-synthase (trpC) terminator⁵

The vector also carries the beta-lactamase gene (bla) and E. colireplication origin from plasmid pUC 18⁶. The detailed plasmid map isprovided in FIG. 3.

C1 protoplasts were transformed with plasmid pUT970G following the sameprocedure already described in example 1. The fusion protein (Shble::GAM hinge::HLZ) is functional with respect to thephleomycin-resistance thus allowing easy selection of the C1transformants. Moreover, the level of phleomycin resistance correlatesroughly with the level of hlz expression.

The HLZ production of C1 transformants (phleomycin-resistant clones) wasanalysed by lysozyme-activity assay as follow: Primary transformantswere toothpicked to GS+phleomycin (5 μg/ml) plates (resistanceverification) and also on LYSO plates (HLZ activity detection byclearing zone visualisation.sup.1, 10). Plates were grown for 5 days at32° C. Each validated clone was subcloned onto LYSO plates. Twosubclones per transformant were used to inoculate PDA plates in order toget spores for liquid culture initiation. The liquid cultures in IC1were grown 5 days at 27° C. (shaking 180 rpm). Then, the cultures werecentrifuged (5000 g, 10 min.). From these samples, lysozyme activity wasmeasured according to Morsky et al.¹³

TABLE G Active HLZ production levels in Cl Active HLZ concentration inculture media Untransformed NG7C-19 0 mg/1 NG7C-19::970G clone 4 4 mg/1NG7C-19::970G clone 5 11 mg/1  Untransformed UV18-25 0 mg/1UV18-25::970G clone 1 8 mg/1 UV18-25::970G clone 2 4 mg/1 UV18-25::970Gclone 3 2 mg/1 UV18-25::970G clone 2 2.5 mg/1  

These data show that:

1) Points 1 & 2 from example 1 are confirmed.2) Sh ble is functional in Chrysosporium as resistance-marker.3) Sh ble is functional in Chrysosporium as carrier-protein.4) The KEX2-like protease cleavage site is functional in Chrysosporium(otherwise HLZ wouldn't be active).5) Chrysosporium can be used as host for the secretion of a heterologousmammalian protein.[3] C1 Secretion of Trichoderma reesei Xylanase II (XYN2).

C1 strain UV18-25 has been transformed by the plasmids pUT1064 andpUT1065.

pUT1064 presents the two following fungal expression cassettes:

The first cassette allows the selection of phleomycin-resistanttransformants:

-   -   Neurospora crassa cross-pathway control gene 1 (cpc-1)        promoter¹⁴    -   Streptoalloteichus hindustanus phleomycin-resistance gene Sh        ble⁴    -   Aspergillus nidulans tryptophan-synthase (trpC) terminator⁵        The second cassette is the xylanase production cassette:    -   T. reesei_strain TR2 cbh1 promoter¹⁵    -   T. reesei_strain TR2 xyn2 gene (including its signal sequence)¹⁶    -   T. reesei_strain TR2 cbh1 terminator¹⁵

The vector also carries an E. coli replication origin from plasmidpUC19⁶. The plasmid detailed map is provided in FIG. 4.

pUT1065 presents the following fungal expression cassette:

-   -   A. nidulans_glyceraldehyde-3-phosphate dehydrogenase (gpdA)        promoter²    -   A synthetic T. _(—) reesei cellobiohydrolase I (cbh1) signal        sequence^(1,3)    -   S. hindustanus phleomycin-resistance gene Sh ble⁴ used as        carrier-protein¹⁰    -   A linker peptide (SGERK) featuring a KEX2-like protease cleavage        site¹    -   T. reesei_strain TR2xyn2 gene (without signal sequence)¹⁶    -   A. nidulans tryptophan-synthase (trpC) terminator⁵

The vector also carries the beta-lactamase gene (bla) and an E. colireplication origin from plasmid pUC 18⁶. The plasmid detailed map isprovided in FIG. 5.

C1 protoplasts were transformed with plasmid pUT1064 or pUT1065following the same procedure already described in example 1. The fusionprotein in plasmid pUT1065 (Sh ble::XYN2) is functional with respect tothe phleomycin-resistance thus allowing easy selection of the C1transformants. Moreover, the level of phleomycin resistance correlatesroughly with the level of xyn2 expression. In pUT1064, xyn2 was clonedwith its own signal sequence.

The xylanase production of C1 transformants (phleomycin-resistantclones) was analysed by xylanase-activity assay as follow: Primarytransformants were toothpicked to GS+phleomycin (5 μg/ml) plates(resistance verification) and also on XYLAN plates (xylanase activitydetection by clearing zone visualisation.sup.17). Plates were grown for5 days at 32° C. Each validated clone was subcloned onto XYLAN plates.Two subclones per transformant were used to inoculate PDA plates inorder to get spores for liquid culture initiation. The liquid culturesin IC1+5 g/l KPhtalate were grown 5 days at 27° C. (shaking 180 rpm).Then, the cultures were centrifuged (5000 g, 10 min.). From thesesamples, xylanase activity was measured by DNS Technique according toMiller et al.sup.18

TABLE H Active XYN2 production levels in Cl (best producers) Activexylanase II Xylanase II concentration specific activity in culture mediain culture media Untransformed UV18-25 3.9 U./ml 3.8 U./mg total prot.UV18-25::1064 clone 7-1 4.7 U./ml 4.7 U./mg total prot. UV18-25::1064clone 7-2 4.4 U./ml 4.3 U./mg total prot. UV18-25::1065 clone 1-1 29.7U./ml  25.6 U./mg total prot.  UV18-25::1065 clone 1-2 30.8 U./ml  39.4U./mg total prot. 

These data show that:

1) Points 1 to 4 from example 2 are confirmed.2) C1 can be used as host for the secretion of a heterologous fungalprotein.

[4] We also illustrate data from expression of transformed UV18-25wherein the table I shows the results for the plasmids with whichtransformation was carried out. The Table shows good expression levelsfor endoglucanase and cellobiohydrolase using heterologous expressionregulating sequences and signal sequences but also with homologousexpression regulating sequences and signal sequences. The details of thevarious plasmids can be derived elsewhere in the description and fromthe figures. The production occurs at alkaline pH at a temperature of35° C.

TABLE I Expression data of transformed UV18-25 strain Total proteinsCMCase 13-glucanase Culture mg/ml u/ml u/mg u/ml u/mg pH value *UV 18-25100% 100% 100% 100% 100% 7.90 1150-23 94% 105% 111% 140% 149% 7.90 -3096% 105% 110% 145% 151% 8.10 1152-3 94% 112% 120% 147% 156% 7.85 -4 100%105% 105% 132% 132% 7.90 1160-2 69% 81% 118% 90% 131% 7.90 -4 73% 72%98% 83% 114% 8.35 -1 92% 95% 103% 120% 130% 8.45 1162-1 102% 105% 103%145% 142% 8.20 -11 112% 109% 98% 115% 103% 8.20 F6g-20 104% 102% 98%130% 125% 7.90 -25 — — — — — — Culture conditions (shake flask): 88 h,35° C. 230 rpm *all above figures are in relative % to parent UV18-25strain

Appendix to the Examples: Media

Transformation Media:

Mandels Base: MnP Medium: KH2P04 2.0 g/l Mandels Base with (NH4)2SO4 1.4g/l Peptone 1 g/l MgS04.71420 0.3 g/l MES 2 g/l CaCl₂ 0.3 g/l Sucrose100 g/l Oligoelements 1.0 ml/l Adjust pH to 5 MnR MnP CA²⁺: MnP +sucrose 130 g/l MnP Medium + Yeast extract 2.5 g/l CaCl₂2H₂050 mMGlucose 2.5 g/l Adjust pH to 6.5 Agar 15 g/l MnR Soft: MnR with only 7.5g/l of agar. MPC: CaCl₂ 50 mM pH 5.8 MOPS 10 mM PEG 40%

For Selection and Culture

GS: Glucose 10 g/1 Biosoyase  5 g/1 [Merieux] Agar 15 g/1 pH should be6.8 PDA: Potato Dextrose Agar 39 g/1 [Difco] pH should be 5.5 MPG:Mandels Base with K.Phtalate  5 g/1 Glucose 30 g/1 Yeast extract  5 g/1

The regeneration media (MnR) supplemented with 50 μg/ml phleomycin or100-150 μg/ml hygromycin is used to select transformants. GS medium,supplemented with 5 μg/ml phleomycin is used to confirm antibioticresistance.

PDA is a complete medium for fast growth and good sporulation. Liquidmedia are inoculated with 1/20th of spore suspension (all spores fromone 90 mm PDA plate in 5 ml 0.1% Tween). Such cultures are grown at 27°C. in shake flasks (200 rpm).

Isolation and characterisation of C1 proteins

The process for obtaining various proteins is described as are a numberof characteristics of the proteins. Tables A and B and FIG. 36 providedetails of purification scheme and activities. Isolation occurs from theChrysosporium culture filtrate using DEAE-Toyopearl ion exchangechromatography analogously to the method described in WO 98/15633, whichis incorporated herein by reference. The non-bound fraction (F 60-31 CF)obtained from this chromatography was purified using Macro Prep QTM™ ionexchange chromatography after equilibration to pH 7,6. The non-boundfraction (NB) was pooled and bound proteins were eluted in 0-1 M NaClgradient. The NB fraction provided major protein bands of 19, 30, 35 and46 kD and a minor one of 51 kD. In 0-1 M NaCl gradient protein peakswere eluted from various fractions. 39-41 included 28, 36 and 60 kDproteins, 44-48 included 28, 45 and 66 kD as major protein bands with33, 36, 55, 60 and 67 kD proteins, the 49-51 fraction gave 30, 36, 56and 68 kD proteins and the 52-59 fraction included major 33 and 55 kDproteins and minor 28 and 36 kD proteins. The pooled NB fraction wasfurther purified by hydrophobic chromatography on Phenyl Superose™. TheNB fraction was equilibrated with 0.03M Na-phosphate buffer pH 7.0containing 1.2 M (NH4)2SO4 and applied to a column. Adsorbed proteinswere eluted in 1.2-0.6 M (NH4)2SO4 gradient. Thus homogeneous xylanasewith MW 30 and 51 kD and pI 9.1 and 8.7 respectively were obtained aswas a 30 kD protease with pI 8,9.

The xylanases did not possess MUF cellobiase activity and are thus truexylanases. The alkaline 30 kD xylanase (pI 9.1) possessed high activitywithin a very broad pH range from 5-8 maintaining 65% of maximumactivity at pH 9-10; it is a member of the xylanase F family; itspartial nucleotide and amino acid sequences are depicted in SEQ ID No.7. The partial amino acid sequence depicted corresponds to about aminoacids 50-170 from the N terminus of the mature protein. Xylanasesaccording to invention have at least 60%, preferably at least 70%, mostpreferably at least 80% sequence identity of the partial amino acidsequence of SEQ ID No. 7. The corresponding xylanase promoter, which isa preferred embodiment of the invention, can be identified using thepartial nucleotide sequence of SEQ ID No. 7. The 51 kD xylanase (pI 8.7)possessed maximum activity at pH 6 and retained at least 70% of itsactivity at pH 7.5 and it retained at least 50% of its activity at pH8.0. It was not very stable with only 15% activity at pH 5.5 and 4% atpH 7.5. The Michaelis constant toward birch xylan was 4.2 g/l for 30 kDxylanase and 3.4 g/l for 51 kD xylanase. Temperature optimum was highand equal to 70° C. for both xylanases.

The 30 kD protease activity measured towards proteins of the NB fractionappeared to be equal to 0.4.times.10-3 units/ml at 50° C. and pH 7.90kD. The fraction exhibited activity toward dyed casein of 0.4 arbitraryunits/mg (pH 7). Addition of urea as chaotropic agent resulted in 2-3times increase of protease activity. The effect of the protease onxylanase activity was significant. Only 30% xylanase activity remainedat pH 10.3 and 50° C. after 30 minutes of incubation. At pH 8 95% of thexylanase activity remained. Linear alkylbenzene sulfonate (LAS) additionresulted in a dramatic decrease of xylanase activity at pH 8 and 10.3with only 50% xylanase activity after 10 minutes of incubation with orwithout protease inhibitor PMSF. The 30 kD protease was alkaline with pHoptimum at pH 10-11. The activity is inhibited by phenylmethylsulfonylfluoride (PMSF) and not by iodoacetic acid, pepstatin A and EDTA whichcharacterises it as a serine type protease. The protease is not activetowards C1 proteins at neutral pH and 50° C. without chaotropic agents.Increase of pH and the addition of chaotropic agents such as LAS, SDSand urea significantly increase proteolysis.

The 39-41 fraction was purified by hydrophobic chromatography on phenylsuperose. Fractions were equilibrated with 0.03M Na phosphate buffer pH7.2 containing 1.5M (NH4)2SO4 and applied to a column. Adsorbed proteinswere eluted in 1.5-0 M (NH4)2SO4 gradient. Thus homogenous xylanase withMW 60 kD and pI 4.7 was obtained. This xylanase possessed activitiestowards xylan, MUF-cellobioside, MUF-xyloside and MUF-lactoside. Thisxylanase probably belongs to family 10 (family F). This xylanase wasstable at pH from 5 to 8 during 24 hours and retained more than 80%activity at 50° C. It retained 70% activity at pH 5-7 at 60° C. It kept80% activity during 5 hours and 35% during 24 hours at 50° C. and pH 9.At pH 10 60% activity was retained at 50° C. and 0.5 hours ofincubation. After 5 hours of incubation at pH 8 and 60° C. 45% activitywas found decreasing to 0 after 24 hours. It had a pH optimum within thepH range of 6-7 and kept 70% activity at pH 9 and 50% of its activity atpH 9.5. The Michaelis constant toward birch xylan was 0.5 g/l.Temperature optimum was high and equal to 80° C.

Fraction 44-48 was then purified by chromatofocusing on Mono P. A pHgradient from 7.63-5.96 was used for the elution of the proteins. As aresult 45 kD endoglucanase was isolated with a pI of 6. The 45 kD endohad maximum activity at pH 5 toward CMC and at pH 5-7 toward RBB-CMC.The 45 kD endo retained 70% of its maximal activity toward CMC at pH 6.5and 70% of its maximal activity toward RBB-CMC was retained at pH 7.0;50% of its maximal activity toward CMC was retained at pH 7 and 50% ofits maximal activity toward RBB-CMC was retained at pH 8. The Michaelisconstant toward CMC was 4.8 g/l. Temperature optimum was high and equalto 80° C. Other proteins 28, 33, 36, 55, 60 and 66 kD were eluted mixedtogether.

Fraction 52-58 was purified by chromatofocusing on Mono P too with a pHgradient 7.6-4.5. Individual 55 kD endoglucanase with pI 4.9 wasobtained. The 55 kD endo was neutral. It has a broad pH optimum from4.5-6 and 70% activity was retained at pH 7.0 both for CMC and RBB-CMCand 50% activity was retained at pH 8 for both CMC and RBB-CMC. TheMichaelis constant toward CMC was 1 g/l. Temperature optimum was highand equal to 80° C. A number of fractions also held proteins with MW of28, 33 and 36 kD.

45, 48 and 100 kD proteins were isolated from bound DEAE Toyopearlfraction of F 60-8 UF conc of Chrysosporium culture from fractions 50-53using Macro Prep Q chromatography.

Fraction 50-53 was equilibrated with 0.03 M imidazole HCL buffer, pH5.75 and was applied to a column and the adsorbed proteins were elutedin 0.1-0.25 M NaCl gradient for 4 h. As a result 45 kD (pI 4.2), 48 kD(pI 4.4) and 100 kD (pI 4.5) proteins were isolated in homogenousstates.

The 45 kD protein is supposedly an alpha, beta-galactosidase by virtueof its activity toward p-nitrophenyl alpha-galactoside and p-nitrophenylbeta-galactoside. The pH optimum was 4.5 70% activity was maintained atpH 5.7 and 50% of its activity was retained at pH 6.8. The temperatureoptimum was 60° C.

The 48 kD protein was a cellobiohydrolase having high activity towardp-nitrophenyl beta-glucoside and also activities toward MUFcellobioside, MUF lactoside and p-nitrophenyl butyrate. The 48 kDprotein had a pH optimum of 5 toward CMC and 5-6 toward RBB-CMC.

The 100 kD protein with pI 4.5 possessed activity only towardp-nitrophenyl butyrate. It is probably an esterase but is not a feruloylesterase as it had no activity against the methyl ester of ferulic acid.It had neutral/alkaline pH optimum (pH 8-9) and optimal temperature of55-60° C.

The 90 kD protease with pI 4.2 was isolated from the bound fraction andthe activity measured towards proteins of the NB fraction appeared to beequal to 12.times.10-3 units/ml at 50° C. and pH 7.90 kD. The fractionexhibited activity toward dyed casein of 0.01 arbitrary units/mg (pH 7).Addition of urea as chaotropic agent resulted in 2-3 fold increase ofprotease activity as did addition of LAS at both pH 7 and 9 (50° C.).The 90 kD protease was neutral with pH optimum at pH 8. The activity isinhibited by phenylmethylsulfonyl fluoride (PMSF) and not by iodoaceticacid, pepstatin A and EDTA which characterises it as a serine typeprotease.

Also isolated from the bound fraction were 43 kD endoglucanase with pI4.2 (fraction 33-37) and 25 kD endoglucanase with pI 4.1 (fraction39-43), 55 kD cellobiohydrolase with pI 4.9 (fraction 39-43) and 65 kDpolygalacturonase with pI 4.4 (fraction 39-43). The endoglucanases didnot possess activity towards avicel or MUF cellobioside and possessedhigh activity toward MC, RBB-CMC, CMC41, beta-glucan and endoglucanase.The 25 kD endo did not produce glucose from CMC and the 43 kD endo did.No glucose was formed from avicel. The pH optimum for the 43 kD proteinwas 4.5 with 70% maximum activity maintained at pH 7.2 and 50% at pH 8.The 43 kD endo kept 70% activity at pH 5 and 6 during 25 hours ofincubation. It kept only 10% at pH 7 during this incubation period. The25 kD endo had pH optimum of activity at pH 5 toward CMC and broad pHoptimum of activity toward RBB-CMC with 70% of the maximum activitybeing kept at pH 9 and with 50% of the maximum activity being at pH 10.It kept 100% activity at pH 5 and 6 and 80% at pH 7, 8, 8.6 and 9.6during 120 hours of incubation. The 25 kD endo had a temperature optimumof activity at 70° C. The 43 kD endo had a temperature optimum ofactivity at 60° C. The Michaelis constants towards CMC were 62 and 12.7g/l for 25 and 43 kD endo respectively. The polygalacturonase is apectinase. The Michaelis constant toward PGA was 3.8 g/l. The pH optimumof PGU activity is within pH range 5-7 and T optimum within 50-65° C.

Genes encoding C. lucknowense proteins were obtained using PCR andcharacterised by sequence analysis. The corresponding full genes wereobtained by screening (partial) gene libraries using the isolated PCRfragments. The full gene of the 43 kD endoglucanase (EG6, Family 6) ofthe C1 strain has been cloned, sequenced and analysed (including 2.5 kbpromoter region and 0.5 kb terminator region). Its nucleotide and aminoacid sequences are depicted in SEQ ID No. 6. Predicted molecular weightof the mature protein is 39,427 Da and predicted pI is 4.53, whichvalues correspond well with the measured values. Protein alignmentanalysis with other glycosyl hydrolases of the family 6.2 shows thatC1-EG6 does not include a cellulose-binding domain (CBD) Homologyanalysis using SwissProt SAMBA software (Smith & Waterman algorithm, Gappenalty 12/2, alignment 10, Blosum62 matrix) shows that C.sub.1-EG6 has51.6% identity with Fusarium oxysporum EG-B (over 376 amino acids),51.0% identity with Agaricus bisporis CBH3 (over 353 amino acids), and50.7% identity with Trichoderma reesei CBH2 (over 367 amino acids). Theputative signal sequence runs Met 1 to Arg 28. The promoter containsseveral potential CreA binding sites, so it is very likely that thispromoter would be subject to glucose repression in a fungal strain withworking CreA regulation.

Similarly, the full gene of the 25 kD endoglucanase (EG5, Family 45) ofthe C1 strain has been cloned, sequenced and analysed (including 3.3 kbpromoter region and 0.7 kb terminator region). The nucleotide and aminoacid sequences are depicted in SEQ ID No. 5. Predicted molecular weightof the mature protein is 21,858 Da and predicted pI is 4.66, whichvalues correspond well with the measured values. This is the smallestfungal endoglucanase known to date. Protein alignment analysis withother glycosyl hydrolases of the family 45 shows that C1-EG5 does notinclude a cellulose-binding domain (CBD), nor a cohesin/dockerin domain.Homology analysis using NCBI-BLASTP2 software (Gap penalty 11/1,alignment 10, Blosum62 matrix) shows that the closest homologous proteinto C1-EG5 is Fusarium oxysporum EG-K with 63% identity. The putativesignal sequence runs Met 1 to Ala 18. The promoter contains manypotential CreA binding sites, so it is very likely that this promoterwould be subject to glucose repression in a fungal strain with workingCreA regulation.

Furthermore, an additional endoglucanase was found by PCR based onfamily 12 cellulases homology analysis. The partial nucleotide and aminoacid sequence of this additional endoglucanase (EG3, Family 12) is givenin SEQ ID No. 8.

The 55 kD protein was a cellobiohydrolase (referred to herein as CBH1)with activity against MUF-cellobioside, MUF lactoside, filter paper andavicel, also against p-nitrophenyl β-glucoside, cellobiose andp-nitrophenyl lactoside. Its activity toward MUF cellobioside isinhibited by cellobiose. The inhibition constant 0.4 mM was determined.The Michaelis constant toward MUF cellobioside was 0.14 mM, toward MUFlactoside was 4 mM and toward CMC was 3.6 g/l. The pH optimum is ratherbroad from 4.5 to 7.50% of maximum activity toward CMC and 80% activitytoward RBB-CMC is kept at pH 8. 70-80% activity within pH 5-8 is keptduring 25 hours of incubation. The temperature optimum is 60-70° C. CBH1is probably a member of the cellobiohydrolase family 7; its partialnucleotide and amino acid sequences are depicted in SEQ ID No. 9. Thepartial amino acid sequence depicted corresponds to about amino acids300-450 from the N terminus of the mature protein. A cellobiohydrolaseaccording to the invention has at least 60%, preferably at least 70%,most preferably at least 80% sequence identity of the partial amino acidsequence of SEQ ID No. 9. The corresponding CBH promoter, which is apreferred embodiment of the invention, can be identified using thepartial nucleotide sequence of SEQ ID No. 9. A synergistic effect wasobserved between 25 kD endo and 55 kD CBH during avicel hydrolysis.Synergism coefficient was maximal at the ratio of 25 kD endo to 55 kDCBH 80:20. The K.sub.syn was 1.3 at its maximum.

The expression level of five main Chrysosporium genes was studied byNorthern analysis. Various strains of C. lucknowense were grown in richmedium containing Pharmamedia with cellulose and lactose (medium 1) orrich medium containing Pharmamedia and glucose (medium 2) at 33° C.After 48 h, mycelium was harvested and RNA was isolated. The RNA washybridised with 5 different probes: EG5, EG6, EG3, Xy1F and CBH. Afterexposure, the Northern blots were stripped and hybridised again with aprobe for ribosomal L3 as a control for the amount of mRNA on the blot.Most strains showed very high response for CBH and high response forXy1F in medium 1; in medium 2, half of the strain showed high responsefor all genes, and the other half showed low response. The order ofexpression strength was deduced from these data as CBH>Xy1F>EG5>EG3>EG6.

Tables A and B and FIG. 36 illustrate the details of the above.

Advanced Isolation and Characterisation of C1 Genes and Gene ExpressionSequences of CBH1, XYL1, EG3 and GPD Construction of a BlueSTAR GeneLibrary of UV18-25

Chromosomal DNA of UV18-25 was partially digested with Sau3A, fragmentsof 12-15 kb were isolated and ligated in a BamHI site of cloning vectorBlueSTAR. Packaging of 20% of the ligation mixture resulted in a genelibrary of 4.6.times.10.sup.4 independent clones. This library wasmultiplied and stored at 4° C. and −80° C. The rest of the ligationmixture was also stored at 4° C.

Screening the Gene Library of UV18-25 for Isolation of the Genes forCbh1, Eg3, Xyl1 and Gpd1

For the isolation of the different genes, in ±−.7.5×10.sup.4 individualBlueSTAR phages per probe were hybridized in duplicate. Hybridisationwas carried out with the PCR fragments of cbh1, eg3 and xyl1 (asdescribed in PCT/NL99/00618) at homologous conditions (65° C.; 0.2×SSC)and with the gpd1 gene of A. niger at heterologous conditions (53° C.;0.5×SSC). The number of positive signals is given in Table K. Thepositive clones were rescreened and for each clone two individual phageswere used for further experiments. DNA of the different clones wasanalysed by restriction analysis to determine the number of differentclones isolated from each gene (results are given in Table K).

As for each of the 4 genes, 4-6 different clones were isolated, weconclude that the primary gene library (±−0.4−5×10⁴ clones) representsabout 5× genome of UV18-25. From this result we conclude that thecomplete genome of UV18-25 is represented in 9×10³ clones. Based on anaverage genomic insert of 13 kb, this would indicate a genome size of±−120 Mb, which is 3 times the size of the Aspergillus genome.

PCR reactions with specific primers for the gene present on the plasmid(based on previous sequence determination from the isolated PCRfragments) and the T7 and T3 primer present in the polylinker ofpBlueSTAR we were able to determine the location of the genes in anumber of clones. From each gene a plasmid was used for sequencedetermination of the gene.

Sequence Analysis of the Cloned Genes

For the cbh1, xyl1, eg3 and the gpd1 gene, the results of the sequencedetermination are represented in SEQ ID No's 1, 2, 3 and 4 respectively.Also the deduced amino acid sequences of the proteins are represented inthese SEQ ID No's 1-4. Some properties of the proteins are given inTable L. It should be mentioned that the position of the start of thetranslation and the introns is based on homology with genes from thesame family (i.e. paper genetics).

CBH1

From the amino acid sequences of CBH1, we concluded that the protein isabout 63 kD in size and that a cellulose binding domain (CBD) is presentat the C-terminal part of the protein. Interestingly, no evidence wasfound for the presence of a CBD in the isolated 55 kD major protein.However, the presence of the isolated peptides from this 55 kD majorprotein in the encoded CBH1 protein (SEQ ID No. 1), confirms that the 55kD protein is encoded by the cloned gene. A possible explanation ofthese results is that the 55 kD protein is a truncated version of theCBH1 protein lacking the CBD.

Xyl1

From the amino acid sequences of xyl1 we conclude that also here a CBDis present, in this protein at the N-terminal side. In the literatureonly two more xylanases with a CBD are known (Fusarium oxysporum andNeocallimastix patriciarum). The estimated size of the Xyl1 protein is43 kD and several peptides isolated from a 30 kD xylanase originate fromthis protein (SEQ ID No. 2). It should be noted that a considerablenumber of the isolated peptides could not be found in the encodedsequence. This could indicate that alternative xylanase proteins arepresent in UV 18-25. In previous analysis, no evidence was found for thepresence of CBD in this 30 kD protein. Also from these results wehypothesized that the CBD of the protein is cleaved of by proteolysis.This hypothesis will be analysed further (by determination ofactivities, N-terminal sequences and sizes of the different proteins inthe different C1 strains: C1 wild type, NG7C, UV13-6, UV18-25 andprotease.sup.-mutants of UV18-25) Also the effect of the presence orabsence of the CBD on enzymatic activities has to be analysed in detailfurther. Overexpression of the full length genes in various C1 hosts maybe considered. The presence of a cellulose binding domain (CBD) is aparticular feature of this enzyme; the only other known family 10glycolytic enzyme (xylanase) having a CBD is the Fusarium oxysporumXy1F. The invention thus pertains to fungal xylanases having a CBD otherthan the Fusarium oxysporum xylanase.

EG3

From the amino acid sequence of EG3 it could be concluded that EG3 is afamily 12 protein. The gene encodes a preproprotein with a dibasic (K-R)propeptide processing site. The C1EG3 protein is 62% similar and 54%identical to the B1 EG3 protein. One putative glycosylation site ispresent at the C-terminal part of the protein (SEQ ID No. 3).

Gpd1

The DNA sequence of the C-terminal part of the gpd1 gene is notdetermined, since we are primarily interested in the promoter sequencesof this gene (SEQ ID No. 4).

The proteins XYL1 and EG3 of C. lucknowense are 54-70% identical totheir closest homologue in the Genbank DATABASE (Table L). Notable isthe strong homology of the CBH1 and the EG5 proteins to their relatedHumicola grisea proteins (74-82% identical). Interestingly the closestrelated proteins to the EG6 protein are only 46-48% identical.

Also notable is that in most cases the closest homologues originate fromFusarium, Humicola or other Pyrenomycetous fungi (Table L), whereasChrysosporium belongs to the Plectomycetous fungi according to the NCBItaxonomy database (Table L).

TABLE K Screening of 7.5 × 10⁴ phages of the gene library of UV18-25with PCR fragments of UV18-25 for the cbhl gene, the eg3 gene and thexyll gene (homologous conditions) and with the gpdA gene of A. niger(heterologous conditions). DNA isolation and restriction analysis wasused to determine the number of different clones Positive in firstpositive in clone used for Gene screening rescreening different clonessequencing cbhl 8 7 4 pCBH7 eg3 6 6 4 pEG3-3 xyll 9 6 5 pXy15 gpdl 12 126 pGPD4

TABLE L isolated number glycosidase from of amino related sequencesfamily Cl acids introns remarks (% identity/% homology) CBH1  7 70 kD526 1 CBD Humicola grisea (74/82) 55 kD (63 kD) (CBH1: P15828) Fusariumoxysporum (58/68) (CBH: P46238) Neurospora crassa (60/69) (CBH1: P38676)XYL1 10 30 kD 333 3 CBD Fusarium oxysporum (67/72) (43 kD) (Xy1F:P46239) Penicillium simplissicum (63/72) (Xy1F: P56588) Aspergillusaculeatus (61/70) (Xy1F: 059859) EG3 12 — 247 2 prepro Aspergillusaculeatus (60/ (30 kD + peptide 71) (F1-CMCase: P22669) glycos) Hypocreajecorina (56/73) (EG: BAA20140) Aspergillus kawachii (54/69) (CMCase:Q12679) EG6 6(2) 43 kD 395 2 no CBD Fusarium oxysporum (48/59) (EGLB:P46236) Acremonium cellulolyticus (48/58) (CBHII: BAA74458) Agaricusbisporus (46/59) (CBH3: P49075) EG5 45 25 kD 225 3 no CBD Humicolagrisea (82/91) (EG: BAA74957) Fusarium oxysporum (63/78) (EGL-K: P45699)Humicola grisea (62/78) (EG: BAA74956) GPD1 — — Incomplete  2+? —Podospora anserina (85/89) (GPD: P32637) Neurospora crassa 80/86) (GPD:U67457) Cryphonectria parasitica80185) (GPD: P19089)

REFERENCES

The Contents Hereof are Incorporated

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1. A modified cell of a fungal strain derived from Chrysosporiumlucknowense strain C1 (VKM F-3500 D), now classified as Myceliopthorathermophilum C1, comprising at least one nucleic acid sequence encodingone or more polypeptides, wherein at least one nucleic acid sequenceencoding one or more polypeptides is operably linked to one or morepromoters operable in a fungal cell; wherein zero or more of saidnucleic acid sequences encoding one or more polypeptides comprise asequence encoding a signal peptide fused in frame to a sequence encodinga polypeptide; wherein said modified strain expresses one or more ofsaid polypeptides at a higher level than a corresponding parental ornon-modified strain under the same culture conditions.
 2. The modifiedcell of a fungal strain of claim 1, wherein said mutant is obtained by amethod comprising introduction of at least one nucleic acid into a cellof said strain, wherein at least one nucleic acid comprises at least oneheterologous sequence selected from the group consisting of a sequencecomprising a promoter, a sequence encoding a signal peptide, and asequence encoding a polypeptide.
 3. The modified cell of a fungal strainof claim 2, wherein said polypeptide is a heterologous polypeptide ofplant, animal, algal, bacterial, archaebacterial, or fungal origin. 4.The modified cell of a fungal strain of claim 1, wherein saidpolypeptide is a homologous fungal polypeptide.
 5. The modified cell ofa fungal strain of claim 1, wherein said polypeptide is selected fromthe group consisting of carbohydrate-degrading enzymes, proteases,lipases, esterases, hydrolases, oxidoreductases, and transferases. 6.The modified cell of a fungal strain of claim 1, wherein saidpolypeptide is selected from the group consisting of fungal enzymesallowing production or over production of primary metabolites, includingorganic acids, and secondary metabolites, including antibiotics.
 7. Themodified cell of a fungal strain of claim 1, wherein said polypeptide isinactivated at a pH below
 6. 8. The modified cell of a fungal strain ofclaim 1, wherein said polypeptide is an enzyme that retains 70% or moreof its activity in a buffered composition having a pH above
 6. 9. Themodified cell of a fungal strain of according to claim 2, at least onenucleic acid comprises a sequence encoding a heterologous signalpeptide.
 10. The modified cell of a fungal strain of claim 1, whereinsaid signal peptide is a fungal signal peptide.
 11. The modified cell ofa fungal strain of claim 10, wherein the fungal signal peptide is asignal peptide obtained from a fungal polypeptide selected from thegroup consisting of a cellulase, a β-galactosidase, a xylanase, apectinase, an esterase, a protease, an amylase, a polygalacturonase, anda hydrophobin.
 12. The modified cell of a fungal strain of claim 1,wherein said nucleic acid further comprises a sequence encoding apolypeptide conferring a selectable or screenable phenotype to saidcell.
 13. The modified cell of a fungal strain of claim 12, wherein saidphenotype is a selectable marker that confers resistance to a drug orrelieves a nutritional defect.
 14. The modified cell of a fungal strainof claim 1, wherein said nucleic acid comprises one or more heterologouspromoters.
 15. The modified cell of a fungal strain of claim 1, whereinsaid nucleic acid comprises one or more fungal promoters.
 16. Themodified cell of a fungal strain claim 15, wherein said promoter is aninducible promoter.
 17. The modified cell of a fungal strain ofaccording to claim 15, wherein said promoter is a highly-transcribedpromoter.
 18. The modified cell of a fungal strain of claim 1, whereinsaid mutant is obtained by a method of mutagenesis involving one or moresteps selected from the group consisting of UV irradiation and chemicalmutagenesis.
 19. The modified cell of a fungal strain of claim 18,wherein the method of mutagenesis comprises the steps of: (a) treatmentwith UV irradiation; (b) treatment withN-methyl-N′-nitro-N-nitrosoguanidine, and (c) treatment with UVirradiation.
 20. The modified cell of a fungal strain of claim 1,wherein said fungal strain is a mutant derived from Chrysosporiumlucknowense strain C1 (VKM F-3500 D), now classified as Myceliopthorathermophilum C1, selected from the group consisting of C1 UV13-6 mutantdeposited as accession number VKM F-3632 D, with a deposit date of Sep.20, 1998, C1 NG7C-19 mutant deposited as accession number VKM F-3633 D,with a deposit date of Sep. 20, 1998, and C1 UV18-25 mutant deposited asaccession number VKM F-3631 D, with a deposit date of Sep. 2, 1998.